Phase 3 Trial of Recombinant Human Alkaline Phosphatase for Patients with Sepsis-Associated Acute Kidney Injury (REVIVAL)

Abstract

Purpose: Ilofotase alfa is a human recombinant alkaline phosphatase with reno-protective effects that showed improved survival and reduced MAKE90 in sepsis-associated acute kidney injury (SA-AKI) patients. ‘REVIVAL’, was aphase 3 trial, conducted to confirm its efficacy and safety.
Methods: In this international double-blinded randomized-controlled trial, SA-AKI patients were enrolled <72 hours on vasopressor and <24 hours of AKI. The primary endpoint was 28-day all-cause mortality. The key secondary endpoint was Major Adverse Kidney Events up to day 90 (MAKE90).
Results: 650 patients were treated and analyzed for safety; and 649 for efficacy data (ilofotase alfa n=330; placebo n=319). The observed mortality rates in the ilofotase alfa and placebo groups were 27.9% and 27.9% (nominal one-sided p-value of 0.50) at 28 days, and 33.9% and 34.8% (p=0.41) at 90 days. The trial was stopped for futility on the primary endpoint. The observed proportion of patients with MAKE90 was 56.7% in the ilofotase alfa group vs. 64.6% in the placebo group (p=0.02), mainly due to the number of patients who received renal replacement therapy (28.2% vs. 36.4%). There was evidence of heterogeneity of treatment effect with a marked reduction in MAKE90 events in patients with pre-existent impaired renal function randomized to ilofotase alfa (p=0.024). Adverse events were reported in 67.9% and 75.0% patients in the ilofotase and placebo group.
Conclusion: Among critically ill patients with SA-AKI, ilofotase alfa did not improve day 28 survival. There may however be reno-protective properties, especially among patients with pre-existing renal disease. No safety concerns were identified.
Trial registration and date of registration: ClinicalTrials.gov number NCT04411472, May-28-2020

Full text

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Phase 3 Trial of Recombinant Human Alkaline
Phosphatase for Patients with Sepsis-Associated
Acute Kidney Injury (REVIVAL)
Peter Pickkers (  Peter.Pickkers@radboudumc.nl )
University Medical Centre St Radboud Nijmegen https://orcid.org/0000-0002-1104-4303
Derek Angus
University of Pittsburgh School of Medicine
Kristie Bass
: D&A Pharma
Rinaldo Bellomo
Austin Hospital
Erik van den Berg
: D&A Pharma
Juliane Bernholz
: D&A Pharma
Morten H Bestle
Copenhagen University Hospital Glostrup: Rigshospitalet Glostrup
Kent Doi
University of Tokyo: Tokyo Daigaku
Christopher Doig
University of Calgary Cumming School of Medicine
Ricard Ferrer
Vall d'Hebron Research Institute: Vall d'Hebron Institut de Recerca
Bruno Francois
INSERM
Henrik Gammelager
Aarhus Universitetshospital
Ulf Goettrup
Zealand University Hospital Koge: Sjaellands Universitetshospital Koge
Eric Hoste
Ghent University: Universiteit Gent
Susanne Iversen
Slagelse Hospital: Slagelse Sygehus
Michael Joannidis

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Medical University of Innsbruck: Medizinische Universitat Innsbruck
John Kellum
Spectral Solutions
Kathleen Liu
University of California San Francisco Department of Medicine
Melanie Meersch
University Hospital Munster: Universitatsklinikum Munster
Ravindra Mehta
UC San Diego: University of California San Diego
Scott Millington
University of Ottawa
Patrick Murray
University College Dublin
Alistair Nichol
University College Dublin
Marlies Ostermann
Guy's and Saint Thomas' Hospitals NHS Trust: Guy's and St Thomas' NHS Foundation Trust
Ville Pettila
University of Helsinki: Helsingin Yliopisto
Christopher Solling
Viborg Regional Hospital: Regionshospitalet Viborg
Matthias Winkel
: D&A Pharma
Paul Young
Wellington Hospital Library: University of Otago Wellington
Alexander Zarbock
University Hospital Munster: Universitatsklinikum Munster
Research Article
Keywords: Sepsis, Acute Kidney Injury, Chronic Kidney Disease, MAKE90
Posted Date: August 2nd, 2023
DOI: https://doi.org/10.21203/rs.3.rs-3210421/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
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Abstract
Purpose: Ilofotase alfa is a human recombinant alkaline phosphatase with reno-protective effects that
showed improved survival and reduced MAKE90 in sepsis-associated acute kidney injury (SA-AKI)
patients. ‘REVIVAL’, was aphase 3 trial, conducted to conrm its ecacy and safety.
Methods: In this international double-blinded randomized-controlled trial, SA-AKI patients were enrolled
<72 hours on vasopressor and <24 hours of AKI. The primary endpoint was 28-day all-cause mortality.
The key secondary endpoint was Major Adverse Kidney Events up to day 90 (MAKE90).
Results: 650 patients were treated and analyzed for safety; and 649 for ecacy data (ilofotase alfa
n=330; placebo n=319). The observed mortality rates in the ilofotase alfa and placebo groups were 27.9%
and 27.9% (nominal one-sided p-value of 0.50) at 28 days, and 33.9% and 34.8% (p=0.41) at 90 days. The
trial was stopped for futility on the primary endpoint. The observed proportion of patients with MAKE90
was 56.7% in the ilofotase alfa group vs. 64.6% in the placebo group (p=0.02), mainly due to the number
of patients who received renal replacement therapy (28.2% vs. 36.4%). There was evidence of
heterogeneity of treatment effect with a marked reduction in MAKE90 events in patients with pre-existent
impaired renal function randomized to ilofotase alfa (p=0.024). Adverse events were reported in 67.9%
and 75.0% patients in the ilofotase and placebo group.
Conclusion: Among critically ill patients with SA-AKI, ilofotase alfa did not improve day 28 survival. There
may however be reno-protective properties, especially among patients with pre-existing renal disease. No
safety concerns were identied.
Trial registration and date of registration: ClinicalTrials.gov number NCT04411472, May-28-2020
Take-home message
Sepsis-associated acute kidney injury in patients admitted to an intensive care unit is associated with
signicant morbidity and mortality. There is currently no pharmaceutical treatment. Although we found
no evidence that ilofotase alfa improved survival, its use reduced major adverse kidney events (mortality,
new onset, RRT, >25% reduction in eGFR, or rehospitalization) up to 90 days. The reduction in such events
was most marked in patient with pre-existent renal impairment.
140-character Tweet
Ilofotase alfa did not reduce mortality but showed a consistent pattern of reno-protection in patients with
sepsis-associated AKI.
Introduction
Sepsis is the leading cause of acute kidney injury (AKI) in critically ill patients as a consequence of
inammatory, direct nephrotoxic, and ischemic processes[1–4]. Development of AKI during sepsis (SA-

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AKI) is independently associated with increased morbidity and mortality[5, 6]. Patients with SA-AKI are at
risk of developing chronic kidney disease (CKD) resulting in a considerable burden for patients and
society[7]. Conversely, underlying CKD markedly increases the risk of AKI and the risk increases
proportionally with severity of CKD[8]. Pre-existent CKD complicated by AKI is common in critically ill
patients and associated with delayed kidney function recovery and increased risk of rehospitalization and
development of end stage renal disease[9].
There are no pharmacological therapies approved for the treatment of SA-AKI. Management consists of
only secondary prevention and supportive care strategies, such as uids and renal replacement therapy
RRT[5, 10]. Alkaline phosphatase (ALP) is an endogenous detoxifying enzyme that plays a signicant role
in host defense and innate immunity, particularly acting as an endogenous anti-inammatory protein[11,
12]. For example, removal of one of two phosphate groups abolishes the biological activity of endotoxin;
the dephosphorylated endotoxin acts as a toll-like receptor 4 (TLR4) antagonist[13]. Furthermore, ALP
dephosphorylates extracellular adenosine triphosphate (ATP), that is pro-inammatory, resulting in
generation of adenosine, which has anti-inammatory and tissue protective effects. In particular, the
kidney is negatively affected by increased levels of ATP while adenosine has reno-protective effects[14].
Hence, dephosphorylation attenuates the inammatory response and exerts tissue-protective
properties[15]. In animal models of sepsis, ALP administration dampens inammation and reduces
mortality[12, 16], but also protects against ischemia-reperfusion injury[17–19].
To augment therapeutic ecacy, human recombinant ALP, named ilofotase alfa, was developed,
consisting of an intestinal ALP sequence (highest biological activity) and a crown domain corresponding
with placental ALP sequence (to enhance stability)[20]. Dephosphorylation properties of ilofotase alfa
were conrmed[20] and considered to reduce systemic and local inammation and attenuate organ
damage[14]. Two small phase 2 studies with bovine ALP[15, 21] demonstrated reduced urinary excretion
of tubular injury markers and more pronounced improvement of endogenous creatinine clearance. In
addition to renal protective effects, a survival benet was observed in a large phase 2 trial[22]. The
REVIVAL study aimed to conrm the effect of ilofotase alfa on 28-day all-cause mortality in critically ill
patients with SA-AKI; major adverse kidney events by 90 days (MAKE90) was the key secondary endpoint.
Materials and methods
Ethics and dissemination
This trial was conducted in accordance with the protocol and consensus ethical principles of
international guidelines including the Declaration of Helsinki, Council for International Organizations of
Medical Sciences (CIOMS) International Ethical Guidelines, and International Conference on
Harmonization (ICH) Good Clinical Practice (GCP) Guidelines. The protocol, the single substantial
protocol amendment and other relevant documents were reviewed and approved by the Institutional
Research Board (IRB) / Institutional Ethics Committee (IEC) in the relevant centers prior to being used in
the trial.

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Trial design
This was a phase 3, multi-center, randomized, double-blind, placebo-controlled, 2-arm parallel-group-
sequential design trial in which patients with SA-AKI were randomly assigned in a 1:1 ratio to ilofotase
alfa or matching placebo in Europe, North America, Australia, New Zealand, and Japan.
Patients, randomization, and study medication
The population studied were adult patients with sepsis and recent onset AKI requiring vasopressor
support. The complete inclusion and exclusion criteria are described in supplemental Table1. Full details
on population and study design (including the protocol amendment) and conduct were previously
published[23]. Briey, three patient cohorts were dened: i) patients with SA-AKI with a pre-AKI reference
estimated glomerular ltration rate (eGFR) ≥ 45 mL/min/1.73 m2 and no proven or suspected COVID-19
at time of randomization (‘main trial population’); ii) patients with a pre-AKI reference eGFR ≥ 25 and < 45
mL/min/1.73 m2 (‘moderate-to-severe CKD population or mCKD’) with sepsis and AKI and no proven or
suspected COVID-19 at time of randomization; iii) patients with COVID-19-induced sepsis and AKI at time
of randomization (‘COVID-19 population’). The ‘all combined population’ includes all three cohorts. The
randomization schedule was stratied by site and modied Sequential Organ Failure Assessment
(mSOFA), excluding the neurological component of SOFA. An independent statistician generated a
permuted block randomization schedule for an interactive voice/web response system, which linked
sequential patient randomization numbers to treatment codes.
Study medication was administered, within 24 hours if sepsis was present prior to AKI or within 48 hours
if AKI was present when sepsis was diagnosed, at 1.6mg (1,000 U) per kg of patient body weight up to
120 kg, with a xed dose of 192mg in patients > 120 kg[23]. Patients received study medication as a 1-
hour infusion once daily for 3 consecutive days. All personnel involved in this study were blinded to
treatment assignment and clinicians were not allowed to measure serum ALP concentrations until day
14[24]. The trial drug was provided in addition to usual care as outlined in the Surviving Sepsis Campaign
guidelines[25] and KDIGO guidelines[5]. Initiation and termination of RRT was based on conventional
criteria[26–28]. A Data Management Committee (DMC) was installed and provided with stopping rules
based on predened threshold for futility or early success.
Primary endpoint
The primary ecacy endpoint was 28-day all-cause mortality.
Secondary endpoints
Renal endpoints
Key secondary endpoint was Major Adverse Kidney Events by day 90 (MAKE90) and its components. Two
denitions of MAKE90 were used. ‘MAKE-on-day-90’ was based on mortality through day 90 or an eGFR
drop of > 25% at day 28
and
day 90 compared to pre-AKI value, or need for RRT
at
day 90; in comparison

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and in accordance with existing data in the literature[29], ‘MAKE-through-day-90’ included any RRT events
through
day 28 or RRT status
at
day 90 or mortality through day 90, or an eGFR drop of > 25% at day 90,
or rehospitalization[30]. Furthermore, to examine the impact of prior renal function on outcome, we
determined the effect of pre-AKI eGFR on MAKE90.
Other secondary endpoints
Additional secondary endpoints were days alive and free of mechanical ventilation (MV), vasopressors or
inotropes; days alive and discharged from Intensive Care Unit (ICU) through day 28; and time to death
through day 90.
Safety Endpoints
Safety endpoints and adverse events (AEs) were monitored until study day 28 (inclusive). All deaths were
recorded up to study day 180.
Data analyses and statistics
Analysis of the primary ecacy endpoint was conducted according to protocol and Statistical Analysis
Plan (SAP) based on the mITT population, dened as all patients in whom drug administration was
started and analyzed according to treatment allocation. A supplementary SAP was established prior to
unblinding of the data during the rst interim analysis for the exploratory analysis of the all combined
population[23]. Safety analyses were conducted in the safety population as dened in the protocol.
For categorical and binary demographic and baseline characteristics, absolute and relative frequencies
are presented; and for continuous variables, the median and inter quartile ranges (IQR) are presented.
For the endpoints 28-day all-cause mortality, 90-day all-cause mortality, ‘MAKE on day 90’ and “MAKE-
through day 90’, observed proportions are presented along with the difference in proportions (i.e. ilofotase
alfa minus placebo), the 95% condence interval for the difference in proportions and the one-sided
nominal p-value. Because the study was stopped early due to futility, estimated (difference in)
proportions and the 95% condence interval and one-sided nominal p-values using the Kaplan-Meier
method are also provided[31]. In addition, for each treatment group, the effect of pre-AKI eGFR on the
predicted probability of a ‘MAKE through day 90’ outcome was investigated using a logistic regression
model which included treatment group, pre-AKI eGFR, and an interaction term for treatment group by pre-
AKI eGFR.
Safety analyses were conducted in the safety population as dened in the protocol. For patients with AEs,
relative and absolute frequencies are presented, and the distribution of the number of patients with
(serious) AEs has been compared using the chi-squared test.
SAS (SAS software version 9.4; SAS Institute Inc., Cary, NC, U.S.A.) was used for all statistical analyses. A
one-sided p-value < 0.025 was considered to indicate statistical signicance. In case the primary was not

Page 7/21
met, further statistical testing was considered explorative and therefore no correction for multiple testing
was performed.
Results
Patient recruitment and demographic characteristics
From November 2020 to July 2022, 649 patients (main [n=567] CKD [n=49] and COVID-19 [n=33] cohorts)
were enrolled and treated in 107 sites in North America, Europe, Japan, and Oceania (Figure 1) and
included in the analyses. Demographic characteristics are presented in Table 1 (demographics per cohort
in Supplemental Table 2). Patient demographics were comparable between the ilofotase alfa and
placebo groups in all three populations and comparable across analysis populations. 
Interim analysis
At the time of the rst interim analysis (based on 411 patients in the main trial population) all-cause 28-
day mortality was 61/208 (29.3%) and 52/203 (25.7%) for the ilofotase alfa and placebo groups,
respectively. The recommendation from the DMC was to stop the REVIVAL trial early based on futility
according to the pre-dened thresholds for predictive probability of success. 
Mortality
For the combined population (including main, CKD, COVID-19 cohorts), there was no difference in
mortality between the ilofotase alfa (92 of 330 patients) and the placebo (89 of 319 patients) groups at
day 28 (27.9% vs 27.9%, respectively). The difference in proportions was -0.02% with a 95% condence
interval of [-6.9%; 6.9%] and a nominal one-sided p-value of 0.50. Day 90 mortality was 112 of 330
patients (33.9%) vs 111 of 319 patients (34.8%). The difference in proportions was -0.86% with a 95%
condence interval of [-8.2%; 6.5%] and a nominal one-sided p-value of 0.41). The Kaplan-Meier analysis
had similar results with nominal one-sided p-values of 0.48 and 0.34 for day 28 and day 90 mortality.
Mortality data per cohort is presented in Supplementary Table 3.
Renal endpoints
There was no difference in observed proportions of patients with a “MAKE on day 90” event between
ilofotase alfa 36.4% (120 of 330) and the placebo 40.1% (128 of 319) groups (difference -3.8% 95%CI
[-11.2%, 3.7%] and a nominal one-sided p-value of 0.16. The estimated proportion of patients with a
‘MAKE-on-day-90’ event was 37.4% versus 42.8% for ilofotase alfa and placebo (difference -5.4%, 95%CI
[-13.2%, 2.4%], with a nominal one-sided p-value of 0.09).
The difference in observed proportions of patients with a ‘MAKE-through-day-90’ event between ilofotase
alfa 56.7% (187 of 330) and the placebo 64.6% (206 of 319) groups was 7.9, 95%CI [-15.4%, -0.4%] and a
nominal one sided p-value of 0.02. The estimated proportion of patients with a ‘MAKE-through-day-90’
event was also lower in the ilofotase alfa group compared to the placebo group (57.2% versus 64.7%,

Page 8/21
respectively (difference -7.4%, 95%CI [-15.2%, 0.4%], with a nominal one-sided p-value of 0.03 (Figure 2).
This effect was predominately driven by the difference in receipt of RRT between the ilofotase alfa and
placebo groups (28.2% vs 36.4%), see Supplementary Table 4.
There was evidence of an interaction between renal function prior to the SA-AKI episode (pre-AKI eGFR)
and the incidence of the ‘MAKE-through-day-90’ event suggesting that the therapeutic ecacy of ilofotase
alfa was more pronounced in patients with a lower pre-AKI eGFR, i.e., more severe pre-existent CKD
(Figure 3).Ilofotase alfa showed a benet over placebo in the probability of a ‘MAKE-through-day-90’
event for patients with pre-AKI eGFR below approximately 90 mL/min/1.73 m2. For each 10 mL/min/1.73
m2 decrease in pre-AKI eGFR, the predicted probability of a ‘MAKE-through-day-90’ event on placebo was
increased by 4% from an average at predicted probability of 0.57 for patients who received ilofotase alfa
(nominal one-sided p-value of 0.024).
Other secondary endpoints
There were no notable differences in the use of other types of organ support, as days alive and free of
non-renal organ support were similar between treatment groups. Also, ICU-LOS was similar, as number of
days alive and discharged from the ICU through day 28were similar between treatment groups
(Supplemental Table 5).
Safety parameters
Reported AEs are summarized by occurrence, percentage of occurrence, severity, seriousness, outcome,
and relation to treatment in the combined patient population (Table 2). A lower number of patients
experienced any AEs in the ilofotase alfa group compared to the placebo group [224/330 (67.9%) in the
ilofotase alfa group and 240/320 (75.0%) in the placebo group with a nominal two-sided p-value based
on the chi-squared test of 0.045]. Overall, the incidence of
serious
AEs was similar between the two
treatment groups. There were no relevant differences observed on SOC (System Organ Class Level)
(Supplemental Table 6).
Discussion
Given the observation that AKI complicates sepsis and patients with AKI have far worse short- and long-
term outcomes, there is high interest in strategies that reduce the incidence or ameliorate the course of
AKI, with the goal of reducing the overall burden on patients. In this phase 3 multi-center, international
double-blind RCT ilofotase alfa did not decrease 28-day all-cause mortality. However, there was evidence
to suggest ilofotase alfa reduced MAKE90 events, mainly driven by lowering the need for RRT in these
patients. Ilofotase alfa was well tolerated, and no safety issues emerged.
The previously demonstrated survival benet of ilofotase alfa[22] was not conrmed in the current trial
and several reasons for this discrepancy with the previous trial in the observed effect of ilofotase alfa on
survival can be put forward. First, the drug might not improve survival and the effect observed in the

Page 9/21
previous phase 2 STOP-AKI trial was a false-positive type 1 error. Second, slight changes in eligibility
criteria between both trials could be responsible. For example, in the REVIVAL trial, patients who already
had AKI when they presented with sepsis were eligible, in contrast to the STOP AKI trial. It is plausible that
the duration of AKI was longer in these patients, possibly limiting the therapeutic ecacy of ilofotase
alfa. It is also plausible that other differences in phenotype between the STOP-AKI and REVIVAL patients
existed that may have been of relevance, e.g. due to an overall change in the ICU population, because of
the COVID-19 pandemic and general heterogeneity in the sepsis population. Indeed, we found evidence of
heterogeneity of treatment effect with a more marked reduction in MAKE90 events in patients with pre-
existent impaired renal function. Third, REVIVAL was conducted in 107 sites worldwide vs 55 sites in
STOP-AKI, which increases generalizability but may have also negatively inuenced therapeutic ecacy.
Although the trial was stopped early because of the lack of an apparent survival benet, ilofotase alfa did
demonstrate reno-protective effects, which is consistent with previous trial results. Earlier clinical data
showed that bovine ALP attenuated urinary excretion of tubular injury marker Glutathione S-transferase
A1 (GSTA1-1), improved creatinine clearance and reduced the need for RRT[15]. Subsequently, a human
recombinant Alkaline Phosphatase (ilofotase alfa) was developed and tested in a phase 2 trial in 301
patients with SA-AKI. Although the primary endpoint in this trial, improvement of endogenous creatinine
clearance (ECC) in 7 days, was not met, longer-term ECC over the 28 day study period was signicantly
better, and in addition, a survival benet and reduction in MAKE-90 events were observed with ilofotase
alfa compared to placebo[22]. In REVIVAL, MAKE90 was markedly reduced in the ilofotase alfa treatment
arm, mainly driven by lower requirement for RRT. Need for RRT is an important component of the
composite MAKE90 endpoint especially due to the strong association with poor outcomes in patients
with sepsis[32–35]. Furthermore, it is recognized that patients with pre-existent CKD are more likely to
suffer from AKI when they develop sepsis and have a higher risk of not recovering renal function[8, 36]. It
is therefore not surprising that patients with a pre-existent lower eGFR, i.e. reduced or no renal reserve, are
more likely to meet a MAKE90 criterion following sepsis and that the reno-protective effect observed with
ilofotase alfa in REVIVAL was more pronounced in these patients. Increased ecacy in patients with pre-
existent CKD has been observed with other interventions as well[8, 27]. As pre-existent renal function was
not collected in the previous (recombinant) alkaline phosphatase trials, the interplay between pre-existent
renal function and therapeutic ecacy of ilofotase alfa is a novel nding that emerged from post-hoc
analysis, and should be interpreted as a hypothesis generating nding that needs to be conrmed.
Strengths of this study include its generalizability, as it was conducted in multiple countries worldwide.
Second, the group sequential trial design allowed for the results to be reported in case the trial was
terminated prematurely for futility. The latter also relates to two limitations. First, in case the primary
endpoint was not met, further statistical testing was considered explorative and therefore no correction
for multiple testing was performed; and second, analyses of combined cohorts were reported to explore
trends based on all enrolled patients. The number of patients enrolled in the COVID-19 and CKD cohorts
were small, and depicted in supplemental les for transparency. Further randomized controlled trials
would be necessary to assess the reno-protective effects of ilofotase alfa.

Page 10/21
Conclusions
The REVIVAL trial was stopped for futility due to lack of evidence of reduced 28-day mortality with
ilofotase alfa treatment in critically ill patients with SA-AKI. Despite this, our ndings were consistent with
preclinical studies and phase 2 trials suggesting reno-protective effects of ilofotase alfa. In particular, we
observed that ilofotase alfa reduced MAKE90, mainly driven by an attenuated need for RRT. The reduction
in MAKE90 with ilofotase alfa appeared most pronounced in patients with a lower eGFR prior to the SA-
AKI episode. A prospective randomized controlled trial is warranted to conrm the benecial renal effects
of ilofotase alfa in patients with SA-AKI and pre-existing CKD.
Declarations
Acknowledgments
PI’s of all enrolling sites and part of the REVIVAL investigators are depicted in supplemental le 1. We
thank all participating patients, their families for their support, as well as study coordinators and research
nurses of all participating sites in delivering the REVIVAL trial. The members of the data monitoring
committee are thanked for their rigorous review of the data throughout the trial process. The authors
thank Danielle Forkink (AM-Pharma, Clinical Trial Coordinator), Mariam Hamed (Phastar Programming
Consultant), Annelies Legters (AM-Pharma, Sr. Director, Global Clinical Trials) and Sharon Richards
(Phastar Biostatistician Consultant) for their support. 
Funding
This work was supported by AM-Pharma. The role of the sponsor in the design of the study was to
coordinate and facilitate processes, where the scientic input was provided by the members of the
protocol committee, steering committee, and specic input by external experts in data management and
statistics. The sponsor contracted an external contract research organization to operationally conduct the
study at the study sites. The contract research organization was responsible for setting up the technical
systems, data collection, quality control, pharmacovigilance, statistics, and further overall management
of the study, under coordination and supervision of the sponsor. The statistical analysis plan was
prepared by the contract research organization with input by principal investigator, sponsor, and external
experts in statistics. The analyses were performed by external contract research organizations. Data were
interpreted by the members of the steering committee, and, in a later phase, all coauthors and external
experts, coordinated by the sponsor, could provide input. The principal investigator was responsible for
preparation of the manuscript. All coauthors reviewed, made adjustments, and approved the manuscript.
The decision to submit the manuscript was made by the principal investigator and other coauthors.
Conict of interests statements:
Peter Pickkers has received travel reimbursements and consulting fees from AM-Pharma in relation to his
role as PI for REVIVAL, and consulting fees from Adrenomed, EBI Paion, Sphingotec, and 4Teen4 outside

Page 11/21
the submitted work.
Derek C. Angus has received consulting fees from AM-Pharma.
Kristie Bass, AM-Pharma BV, The Netherlands.
Rinaldo Bellomo has received consulting fees and research support from AM-Pharma, Baxter, Paion,
Viatris, Jafron Biomedical, and CSL Behring.
Erik van den Berg, AM-Pharma BV, The Netherlands.
Juliane Bernholz, AM-Pharma BV, TheNetherlands.
Morten H. Bestle has received consulting fees from AM-Pharma in relation to his role for REVIVAL and
has conducted contract research for Inotrem outside of the submitted work.
Kent Doi has received consulting fees from AM-Pharma.
Chistopher J. Doig reports no conicts of interest.
Ricard Ferrer hasreceived consulting fees from AM-Pharma.
Bruno Francois has received consulting fees from AM-Pharma as a member of the REVIVAL steering
committee, and consulting fees from Inotrem, Aridis and Enlivex outside the submitted work.
Henrik Gammelagerreports funding from various companies in the form of research grants to (and
administered by) Aarhus University or Aarhus University Hospital. H.G. has received support for attending
meetings by Baxter A/S.
Ulf Goettrup-Pedersen reports no conicts of interest.
Eric Hoste has received a travel grant from AM-Pharma.
Susanne Iversen reports no conicts of interest.
Michael Joannidis has received honoraria or research support from Baxter Healthcare Corp, AM-Pharma,
CLS Behring, Fresenius, Takeda, Sano and Novartis.
John A. Kellum discloses fees paid by AM-Pharma in relation to his role as national PI for REVIVAL and is
currently a full-time employee of Spectral Medical.
Kathleen Liu has been a member of the REVIVAL Steering Committee for AM Pharma. She has been a
consultant/member of the DSMB for Seastar, Novartis, BOA Medical, Baxter, and Biomerieux, and she
holds stock in Amgen.
Melanie Meersch has received lecture fees from Baxter and Fresenius Medical Care.

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Ravindra Mehta reports honoraria for consulting fromBaxter, Biomerieux, Mallinckrodt, GE Healthcare,
Sano, Abiomed, NovaBiomed, Renasym and advisory board reimbursements from AM Pharma, Renibus,
Alexion, Novartis and Guard.
Scott Millington reports no conicts of interest.
Patrick T. Murray has received consulting fees from AM-Pharma (for Clinical Trial Steering Committee
activities), Novartis, Renibus Therapeutics, and Alexion.
Alistair Nichol reports an unrestricted grant from Baxter to support the renal sub study of the TAME trial.
Marlies Ostermann has received speaker honoraria from Fresenius Medical, Baxter and Biomerieux; her
institution received research funding from Baxter, Fresenius Medical, Biomerieux and LaJolla Pharma. 
Christoffer Solling reports no conicts of interest.
Pettila Ville reports no conicts of interest.
Matthias Winkel, AM-Pharma BV, The Netherlands.
Paul J Young has received consulting fees from AM Pharma and from Baxter Healthcare Pty.
Alexander Zarbock has received consulting fees from Astute-Biomerieux, Baxter, Bayer, Novartis, Guard
Therapeutics, AM Pharma, Paion, Renibus, Fresenius, research funding from Astute-Biomerieux,
Fresenius, Baxter, and speakers fees from Astute-Biomerieux, Fresenius, Baxter.
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Tables
Table 1:Demographic and baseline characteristics

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  Combined Patient population
  Ilofotase alfa
(N=330) Placebo
(N=319)
Sex, n (%) Female 124 (37.6 %) 113 (35.4 %)
Male 206 (62.4 %) 206 (64.6 %)
Age, in years  70.0 (62.0,76.0) 70.0 (61.0,76.0)
Weight in kg  84.0 (72.0,100.0) 82.0 (70.0,100.0)
Height in cm  172.0
(164.0,178.0) 170.0
(165.0,178.0)
Body Mass Index in kg/m2 28.9 (25.0,33.2) 27.6 (24.6,34.0)
Mechanical Ventilation Status,
n(%) Off 84 (25.5 %) 74 (23.2 %)
On 246 (74.5 %) 245 (76.8 %)
AKI stage at randomization, n(%) 0 or 1 144 (43.6 %) 129 (40.4 %)
2 93 (28.2 %) 105 (32.9 %)
3 93 (28.2 %) 84 (26.3 %)
Missing  0  1 (0.3 %)
Baseline eGFR  31.2 (21.2,43.3) 30.6 (20.4,42.0)
Pre-AKI eGFR  74.0 (60.1,88.3) 73.9 (59.8,89.0)
mSOFA   9.0 (7.0,10.0)  9.0 (8.0,11.0)
CRP  260.5
(141.2,341.2) 235.0
(155.7,321.3)
Lactate   2.2 (1.5,3.6)  2.4 (1.5,3.7)
PaO2/FiO2 ratio:  225.8
(146.3,304.2) 191.7
(134.0,292.5)
Infection Status Proven 156 (47.3 %) 160 (50.2 %)
Suspected 174 (52.7 %) 159 (49.8 %)
Infection Site: Abdominal 120 (36.4 %) 115 (36.1 %)
CNS   7 (2.1 %)   3 (0.9 %)
Other 32 (9.7 %) 34 (10.7 %)
Pulmonary 97 (29.4 %) 95 (29.8 %)

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  Combined Patient population
  Ilofotase alfa
(N=330) Placebo
(N=319)
Skin or soft-
tissue 24 (7.3 %) 30 (9.4 %)
Unknown 15 (4.5 %) 18 (5.6 %)
Urinary tract 35 (10.6 %) 24 (7.5 %)
Pathogen Proven/Suspected Proven 132 (40.0 %) 126 (39.5 %)
Suspected 192 (58.2 %) 190 (59.6 %)
Missing  6 (1.8 %)  3 (0.9 %)
Type of pathogen: Viral 28 (8.5 %) 18 (5.6 %)
Bacterial 251 (76.1 %) 239 (74.9 %)
 Gram Positive 77 (23.3 %) 67 (21.0 %)
 Gram Negative 92 (27.9 %) 82 (25.7 %)
 Mixed 49 (14.8 %) 53 (16.6 %)
 Missing 33 (10.0 %) 37 (11.6 %)
Missing 51 (15.5 %) 62 (19.4 %)
Note:
- Continuous variable: Median (IQR)
- Patients have been analyzed according to treatment received
Table 2. Overall summary of number of patients with adverse events in the combined patient population

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  Treatment Group
 Ilofotase alfa
(N=330)
n (%)
Placebo
(N=320)
n (%)
P-value*
Adverse events 224 ( 67.9%) 240 ( 75.0%) 0.0446
Serious adverse events 143 ( 43.3%) 141 ( 44.1%) 0.8514
Fatal adverse events 99 ( 30.0%) 98 ( 30.6%) 
Serious non-fatal adverse events 64 ( 19.4%) 62 ( 19.4%) 
Drug-related adverse events 30 ( 9.1%) 32 ( 10.0%) 
Serious drug-related adverse events 16 ( 4.8%) 16 ( 5.0%) 
Adverse events leading to withdrawal of trial drug 9 ( 2.7%) 6 ( 1.9%) 
Serious adverse events leading to withdrawal of trial drug 8 ( 2.4%) 5 ( 1.6%) 
Adverseeventsbyseverity**
Mild 29 ( 8.8%) 31 ( 9.7%) 
Note:
- Patients have been analyzed according to treatment received
* Nominal p-value based on chi-square test
Figures

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Figure 1
Flow chart of the patients participating in the REVIVAL trial.

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Figure 2
Time to rst “MAKE through day 90” event
Proportions of patients without "MAKE through day90”: Ilofotase alfa 42.77%, Placebo 35.33%.
Proportion of patients with “MAKE through day 90” event at Day 90: Ilofotase alfa 57.23%, Placebo
64.67%
The difference (Ilofotase alfa-Placebo) in proportion of patients with “MAKE through day 90” event at Day
90 is -7.44% with a 95% condence interval of (-15.23%, 0.36%).
The nominal one-sided p-value based on the Wald statistic for the difference in proportions is 0.030705
Patients who withdrew prior to day 90 (inclusive) without a “MAKE through day 90” event are censored at
their date of withdrawal.
Patients who withdrew/completed the trial after day 90 were censored at day 90.
Patients who died or were rehospitalized after day 90 are censored at day 90.
Patient with a “Make through day 90” event based on receiving RRT at their Day 90 visit , or a >25% drop
in eGFR at their Day90 visit, are counted as having a “MAKE through day 90” event at day 90.

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Figure 3
Predicted Probability of a “MAKE though day 90” event with 95% Condence Limits
Predicted probabilities are taken from the results of the logistic regression model which included “MAKE
through day 90” as the outcome of interest, and
treatment, pre-AKI reference eGFR, and pre-AKI reference eGFR by treatment interaction (p=0.0235).
Patients who did not meet the criteria for having a conrmed “MAKE through day 90” event are assumed
to not have a “MAKE through day 90” event in the logistic regression model.
Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.
CONSORTchecklist.docx
Supplle1REVIVALSiteprincipleinvestigators.xlsx
SupplementalTableandFiguresREVIVAL.docx