Open access Protocol Study protocol of a randomised, double-­ blind, placebo-­controlled, two-­arm parallel-­group, multi-­centre phase 3 pivotal trial to investigate the efficacy and safety of recombinant human alkaline phosphatase for treatment of patients with sepsis-­associated acute kidney injury Peter Pickkers ‍ ‍,1 Derek C Angus,2 Jacques Arend,3 Rinaldo Bellomo,4,5 Erik van den Berg,3 Juliane Bernholz,3 Morten Bestle,6,7 Kristine Broglio,8 Jan Carlsen,3 Christopher J Doig,9 Ricard Ferrer,10 Michael Joannidis,11 Bruno Francois,12 Kent Doi,13 John A Kellum,14 Pierre-­François Laterre,15 Kathleen Liu,16 Ravindra L Mehta,17 Patrick T Murray ‍ ‍,18 Marlies Ostermann,19 To cite: Pickkers P, Angus DC, Ville Pettilä,20 Sharon Richards,21 Paul Young ‍ ‍,22 Alexander Zarbock ‍ ‍,23 Arend J, et al. Study protocol Anne Louise Kjølbye3 of a randomised, double-­ blind, placebo-­controlled, two-­arm parallel-­group, multi-­centre phase 3 pivotal ABSTRACT trial to investigate the efficacy Introduction Sepsis, the leading cause of acute kidney STRENGTHS AND LIMITATIONS OF THIS STUDY and safety of recombinant injury (AKI), is associated with a high morbidity and ⇒ Apart from the primary endpoint, results of this trial human alkaline phosphatase mortality. Alkaline phosphatase (ALP) is an endogenous may also illustrate the efficacy of ilofotase alfa on for treatment of patients with sepsis-­associated acute detoxifying enzyme. A recombinant human ALP compound, renal endpoints and other organ-­ specific clinical kidney injury. BMJ Open ilofotase alfa, showed no safety or tolerability concerns outcomes. 2023;13:e065613. doi:10.1136/ in a phase 2 trial. Renal function improvement over 28 ⇒ The trial was designed with input from or review by bmjopen-2022-065613 days was significantly greater in the ilofotase alfa group. the US Food and Drug Administration, the European Moreover, a significant relative reduction in 28-­day all-­ Medicines Agency, Japanese Pharmaceuticals and ► Prepublication history and Medical Devices Agency and several national me- additional supplemental material cause mortality of >40% was observed. A follow-­up trial for this paper are available dicinal regulatory authorities in Europe, Australia has been designed to confirm these findings. online. To view these files, and New Zealand. Methods and analysis This is a phase 3, global, multi-­ please visit the journal online ⇒ The group sequential trial design allows for the re- centre, randomised, double-­blind, placebo-­controlled, (http://dx.doi.org/10.1136/​ sults to be reported in case the trial is terminated sequential design trial in which patients are randomly bmjopen-2022-065613). prematurely for futility. assigned to either placebo or 1.6 mg/kg ilofotase alfa. ⇒ In the additional separate COVID-­19 and ‘moderate Received 13 June 2022 Randomisation is stratified by baseline modified Sequential to severe’ chronic kidney disease cohorts, therapeu- Accepted 13 March 2023 Organ Failure Assessment (mSOFA) score and trial site. tic efficacy on clinical endpoints is likely underpow- The primary objective is to confirm the survival benefit ered to reach statistical significance. with ilofotase alfa by demonstrating a reduction in 28-­day © Author(s) (or their all-­cause mortality in patients with sepsis-­associated AKI employer(s)) 2023. Re-­use requiring vasopressors. A maximum of 1400 patients will permitted under CC BY-­NC. No be enrolled at ∼120 sites in Europe, North America, Japan, Ethics and dissemination The trial is approved by commercial re-­use. See rights Australia and New Zealand. Up to four interim analyses relevant institutional review boards/independent ethics and permissions. Published by will take place. Based on predefined decision rules, the committees and is conducted in accordance with the BMJ. trial may be stopped early for futility or for effectiveness. ethical principles of the Declaration of Helsinki, guidelines For numbered affiliations see In addition, patients with COVID-­19 disease and patients of Good Clinical Practice, Code of Federal Regulations and end of article. with ‘moderate to severe’ chronic kidney disease are all other applicable regulations. Results of this study will Correspondence to analysed as 2 separate cohorts of 100 patients each. An determine the potential of ilofotase alfa to reduce mortality Professor Peter Pickkers; independent Data Monitoring Committee evaluates safety in critically ill patients with sepsis-­associated AKI and will ​peter.​pickkers@​radboudumc.​nl data at prespecified intervals throughout the trial. be published in a peer-­reviewed scientific journal. Pickkers P, et al. BMJ Open 2023;13:e065613. doi:10.1136/bmjopen-2022-065613 1 Open access Trial registration number EudraCT CT Number 2019-­0046265-­24. not significantly greater in the treatment group compared US IND Number 117 605 Pre-­results. C ​ linicalTrials.​gov number: with placebo, treatment with ilofotase alfa was associated NCT04411472. with a more pronounced long-­ term improvement of kidney function and lower 28-­day mortality.29 INTRODUCTION Sepsis, defined as a dysregulated host response to infec- Trial objectives tion causing organ dysfunction, is the leading cause of The significant effect on 28-­day mortality observed (as an death worldwide.1 2 Sepsis is also the leading cause of acute exploratory endpoint) in STOP-­AKI requires confirma- kidney injury (AKI).3–5 Patients with sepsis who develop tion in a larger adequately powered, placebo-­controlled, AKI have a higher mortality, while an improved outcome multi-­centre trial. Therefore, the primary objective is observed in patients with resolving AKI.6 7 Patients who of REVIVAL (Recombinant human alkaline phospha- survive a sepsis-­associated AKI episode are at risk of devel- tase SA-­ AKI survival trial) is to confirm or refute the oping chronic kidney disease (CKD), resulting in a high study’s primary endpoint, a reduction in 28-­day all-­cause burden for both the patient and society.8–10 AKI is a multi-­ mortality, in patients with sepsis-­associated AKI treated factorial condition with inflammatory, direct nephrotoxic with 1.6 mg/kg ilofotase alfa. and ischaemic insults combining with other pathogenic responses to rapidly cause dysfunction or failure of the kidney.11–14 Currently, there are no pharmacolog- ical interventions approved for the treatment of sepsis-­ METHODS AND ANALYSIS associated AKI, and renal replacement therapy is the only Patients supportive treatment option available for these patients.15 The target patient population is adult patients in the Alkaline phosphatase (ALP) is an endogenous homod- Intensive Care Unit (ICU) or intermediate care unit imeric enzyme present in many cells and organs (eg, with sepsis requiring vasopressor support and recent intestines, placenta, liver, bone, kidney and granulocytes) onset AKI. Vasopressor support is defined as need for with detoxifying effects through dephosphorylation of ≥0.1 µg/kg/min norepinephrine or equivalent for sepsis-­ endotoxins16 17 and other pro-­inflammatory compounds induced hypotension for at least 1 hour despite adequate such as ATP.18 19 Local ALP concentrations reflect the fluid resuscitation according to clinical judgement. AKI host defence against endotoxin in the kidney.20 During is defined by the KDIGO criteria.30 If the patients are ischaemia, ALP levels are markedly depleted, which is receiving vasopressor support and develops AKI, the associated with the development of AKI.21 Apart from study drug infusion must start within 24 hours following local effects in the kidney, ALP may attenuate the innate the AKI diagnosis. If the patient presents with AKI, the immune response, as dephosphorylation of endotoxin time window is 48 hours. abolishes its biological activity and the dephosphorylated There is no upper age limit for this trial. Consecutive endotoxin acts as a toll-­like receptor 4 antagonist.22 In patients will be systematically screened for AKI as soon animal models of sepsis, ALP administration attenuates as possible following the initiation of vasopressor treat- the inflammatory response and reduces mortality.23 24 ment. Informed consent will be sought in all eligible There is increasing evidence that ALP plays a significant patients with sepsis-­ associated AKI. Inclusion criteria role in host defence and innate immunity, particularly are described in box 1, with the full eligibility criteria in against inflammatory reactions due to endotoxin release.25 online supplemental file 1. The first patient was enrolled on 2 November 2020. If the trial continues to recruit to Recombinant human ALP (recAP and ilofotase alfa) target sample size, it is anticipated to run for 3 years. Ilofotase alfa is a full-­length human chimeric ALP produced by recombinant technology. It is encoded by Trial oversight a human intestinal ALP sequence (highest biological A Trial Steering Committee (TSC) has been established activity)26 wherein the sequence encoding the crown by the Sponsor AM-­Pharma to facilitate design of the domain has been substituted with the corresponding trial, provide leadership and oversight of trial conduct human placental ALP sequence (longest half-­ life).26 in a blinded fashion, and make recommendations to Ilofotase alfa has a mass of approximately 105 kDa based the sponsor. Likewise, a Data Monitoring Committee on the amino acid sequence derived from the DNA (DMC), consisting of independent critical care and sequence and approximately 130 kDa as a fully glyco- nephrology experts not otherwise involved in the trial sylated molecule. and a statistician, has been established to evaluate safety A large phase 2a/2b proof-­of-­concept and dose-­finding data at regular intervals throughout the trial and notify trial (‘STOP-­AKI’, n=301) was conducted with ilofotase the sponsor and the TSC in case of safety concerns that alfa in SA-­AKI after two small phase 2 trials27 28 with lead to a recommendation to stop or modify the trial. The bovine ALP demonstrated attenuation of excretion of DMC will also review the interim analyses reports for effi- tubular injury markers and improvement in renal func- cacy and notify the sponsor and the TSC in case a futility tion. While the improvement in endogenous creatinine or success threshold is reached. The DMC and TSC both clearance over the first week in the STOP-­AKI trial was follow agreed charters. 2 Pickkers P, et al. BMJ Open 2023;13:e065613. doi:10.1136/bmjopen-2022-065613 Open access 3. A COVID-­19 population: patients with proven or suspect- Box 1 Short list of inclusion criteria ed COVID-­19 at time of randomisation with or without Inclusion criteria ‘moderate to severe’ CKD. 1. 18 years or older. Participating sites that formally stated that they would 2. In the ICU or intermediate care unit for clinical reasons. commit to follow the internationally accepted standard 3. Have sepsis requiring vasopressor therapy, that is: of care guidelines for sepsis,31 and those for AKI30 32 were a. Suspected or proven bacterial or viral infection. selected. If new versions of these guidelines are published and during the trial, these will be adopted. b. On vasopressor therapy (≥0.1 µg/kg/min norepinephrine or equiv- A minimum of approximately 450 and a maximum of alent) for sepsis-­induced hypotension for at least 1 hour despite 1400 patients in the main trial population are planned adequate fluid resuscitation according to clinical judgement. to be enrolled at approximately 120 sites predominantly 4. Have AKI according to at least one of the below KDIGO criteria, a across Europe, North America, Japan, Australia and to d: New Zealand. Additionally, approximately 100 patients a. An absolute increase in serum or plasma creatinine (CR) by ≥0.3 mg/dL (≥26.5 µmol/L) within 48 hours. in the ‘moderate to severe’ CKD population and up to or 100 patients in the COVID-­19 population are planned to b. A relative increase in CR to ≥1.5 times the pre-­AKI reference CR be enrolled to generate exploratory data on safety and value, which is known or presumed to have occurred within prior effects of ilofotase alfa in these populations. The final 7 days days. number of patients enrolled will depend on the recom- or mendations of the DMC based on the safety data reviews c. A decrease in urinary output to<0.5 mL/kg/hour for a minimum and interim analyses applying predefined decision rules of 6 hours hours following adequate fluid resuscitation. to determine futility/success. or There will be a maximum of four interim analyses d. If the patient does not have a known history of CKD and there is during the trial and enrolment continues during the no pre-­AKI reference CR value available from the past 12 months: interim analysis. The interim analyses will take place after a CR value ≥1.5 times the age/gender/race adjusted normal val- ue (presented in online supplemental file 1), with the increase in approximately 400, 700, 850 and 1000 evaluable patients CR presumed to have occurred within prior 7 days days. (ie, treated patients in the main trial population who have 5. Provision of signed and dated informed consent form in accordance reached day 28). At the first interim analysis, the trial may with local regulations. be stopped for futility only. At subsequent interim anal- yses, the trial may be stopped for futility, or success (ie, early demonstration of superiority of ilofotase alfa over placebo on 28-­day all-­cause mortality). More details of the Patient and public involvement statement safety data reviews and the interim analyses are provided Reflecting the acute and unpredictable nature of sepsis-­ in the DMC charter and Statistical Analysis Plan (SAP), associated AKI there are no specific patient advocacy which were finalised before the first safety data review. groups at present; closest are organisations that represent The trial includes a pre-­ treatment period, during kidney diseases or sepsis in general and, although they which the patient is screened, baseline assessments are are aware of our programme, they were not specifically performed and the patient is randomised, a treatment consulted on the design of this study. period from day 1 to day 3, during which the patient receives a daily 1 hour continuous intravenous infusion Trial design of 50 mL trial drug (ilofotase alfa 1.6 mg/kg or placebo) This is a phase 3, multi-­centre, randomised, double-­blind, and a follow-­up period from day 4 to day 180, during placebo-­controlled, 2-­arm parallel-­group-­sequential which trial specific assessments and data collection are design pivotal trial in which patients with SA-­ AKI are performed. The trial drug is provided on top of standard randomly assigned in a 1:1 ratio to either ilofotase alfa of care specified by the Surviving Sepsis Campaign guide- or matching placebo. The intended ilofotase alfa dose is lines and KDIGO guidelines. Safety is followed up until 1.6 mg/kg (1000 U) of patient body weight up to 120 kg study day 28 (inclusive). All serious adverse events that (a fixed dose of 192 mg >120 kg) administered as a 1-­hour occur after study day 28 will be reported to the sponsor or infusion once daily for 3 consecutive days. The first infu- designee only if the Investigator considers them possibly, sion is to start as soon as feasible after randomisation. probably or definitely related to the trial drug. All deaths There will be three distinct SA-­AKI trial populations: will be recorded up to study day 180. The trial design 1. The main trial population: patients with sepsis-­associated is presented in figure 1, the timelines for eligibility in AKI and a pre-­AKI reference estimated glomerular fil- figure 2, the trial flow for the individual patient in online tration rate (eGFR) ≥45 mL/min/1.73 m2 and no prov- supplemental figure 1 and the schedule of activities in en or suspected COVID-­19 at time of randomisation. online supplemental file 2. 2. A ‘moderate to severe’ CKD population: patients with sepsis-­ associated AKI a pre-­AKI reference eGFR ≥25 mL/ Statistical considerations min/1.73 m2 and <45 mL/min/1.73 m2 and no proven The primary efficacy endpoint is ‘28-­ day all-­ cause or suspected COVID-­19 at time of randomisation. mortality’, defined as the probability to die (from any Pickkers P, et al. BMJ Open 2023;13:e065613. doi:10.1136/bmjopen-2022-065613 3 Open access Figure 1 Trial design. Up to 1400 patients in the main trial population, up to approximately 100 patients in the ‘moderate to severe’ CKD population and up to approximately 100 patients in the COVID-­19 population will be enrolled. There will be a maximum of four interim analyses, after approximately 400, 700, 850 and 1000 evaluable patients (ie, treated patients in the main trial population who have reached day 28), respectively. At the interim analyses, the trial may be stopped for futility or, from 700 patients onwards, for success (ie, pre-­defined p value for primary endpoint met in the main trial population). Safety will also be assessed at regular intervals and the trial may be stopped or modified for safety concerns1. In the ‘moderate to severe’ CKD population and in the COVID-­19 population, only safety will be assessed at the interim analyses. If the trial is not stopped at one of the interim analyses, a database snapshot will be executed after 1400 patients in the main trial population have reached day 28 to determine if the primary endpoint was met. No further analyses will be performed at this time. An interim lock will take place after all patients in the main trial population have reached day 90. Endpoints defined up to and including day 90 will be analysed and the results used to start the preparation of the CTR. The final DBL will take place after all patients have completed the trial (ie, all patients have completed day 180 or have withdrawn/are lost to follow-­up prior to day 180). If patients in the ‘moderate to severe’ CKD population have not completed the trial at the time of the interim lock at day 90 and/or final DBL at day 180 for patients in the main trial population, a separate interim lock at day 90 and/or final DBL at day 180 may be performed for patients in the ‘moderate to severe’ CKD population in order for the analysis of data in the main trial population to commence. A separate interim lock may also be performed for the COVID-­19 population. 1A full safety review at the time of the 850-­patient interim analysis will only be performed if a futility or success threshold is reached. CKD,chronic kidney disease; CTR,clinical trial report; DBL,database lock; eGFR,estimated glomerular filtration rate; ICF,informed consent form; mSOFA,modified Sequential Organ Failure Assessment score (excluding Glasgow coma score); recAP, recombinant human alkaline phosphatase. cause) up to and including day 28. The primary analysis acceptance of the manuscript, an amended SAP was will be based on a logistic regression model with site as a written and approved prior to the first interim-­analysis. random effect, treatment as a fixed effect, mSOFA score This amendmentment, which were not subjected to peer-­ as an ordered categorical variable and eGFR at baseline as review, is depicted in online supplemental file 4. the single continuous covariate. Analysis of data from each population will also be The primary, secondary and tertiary/explorative performed and presented separately. Formal analyses endpoints are listed in table 1. Summary of statistical (including interim analyses), hypothesis testing and analyses and definitions of endpoints are described in descriptive analyses will be performed on data from online supplemental file 3. the main trial population, whereas descriptive statistics Statistical analyses will be performed by a sponsor-­ (including an estimate of the treatment effect, two-­sided designated statistician, blinded to treatment allocation. 95% CIs and one-­sided p value) will be presented for the Further details of the statistical analyses are described in combined population, ‘moderate to severe’ CKD popula- the SAP, which has been finalised prior to the first safety tion and the COVID-­19 population. review of the trial database (online supplemental file 3). A The Lan-­DeMets approximation of the O’Brien-­Fleming separate DMC SAP describes the analyses to be performed alpha spending function was used to determine the crit- for the safety data reviews and interim analyses. All statis- ical values for declaring success at interim and final anal- tical analyses of efficacy endpoints will be performed on yses. As safety is evaluated by the DMC on a regular basis the modified intention-­to-­treat analysis sets, defined as throughout the trial and the risk of increased mortality, all patients in the population who are randomly assigned that is, inferiority of ilofotase alfa compared with placebo, to trial drug and for whom administration of trial drug is controlled by the futility analyses, the test for thera- was started. All statistical analyses of safety endpoints will peutic efficacy is a superiority test using a one-­sided signif- be performed on the safety analysis sets. Of note, after icance level of 0.025. Online supplemental file 3 shows 4 Pickkers P, et al. BMJ Open 2023;13:e065613. doi:10.1136/bmjopen-2022-065613 Open access treatment group was set at 35% across all scenarios, which was the observed 28-­day mortality in the subgroup of patients with a study baseline eGFR <60 mL/min in STOP-­AKI. In case of an 8% absolute survival benefit of the treatment, this trial has 85.6% power and the mean sample size is expected to be 1010 patients due to the probability of early success or futility. Safety endpoints Safety parameters will be evaluated on the Safety Set. Incidence of (Serious) Adverse Events and Treatment Emergent Adverse Events categorised by MedDRA System Organ Class and Preferred Term will be summarised by trial drug group. Adverse event seriousness, severity, relationship to trial drug and whether leading to discon- Figure 2 Timelines for eligibility. To be eligible for the trial, patients must have both sepsis requiring vasopressor tinuation of trial drug will also be recorded. Anti-­drug therapy and AKI. (1) When AKI is diagnosed before the start antibodies results will be listed, including the results of of vasopressor therapy, infusion of first dose of trial drug the screening test and, if needed, the results of the confir- must start within 48 hours of AKI diagnosis. (2) When AKI matory test and titre determination per dose group. is diagnosed after start of vasopressor therapy, infusion of first dose of trial drug must start within 24 hours of AKI Population PK diagnosis and no more than 72 hours from start of continuous A population pharmacokinetics (PK) analysis of plasma vasopressor therapy for sepsis-­induced hypotension. Start concentration-­time data will be performed using non-­ of AKI is defined as the timepoint where the patient for the linear mixed-­effects modelling. Data from this trial may first time meets any one of the inclusion criteria 4a–4d. Start of vasopressor therapy is defined as the start time of be combined with data from the phase 1 PK, safety and any dose of vasopressor in the first vasopressor treatment tolerability trial in Caucasian healthy adult volunteers,33 period that includes a continuous infusion of ≥0.1 µg/kg/min an additional PK, safety and tolerability trial performed norepinephrine (or equivalent) for sepsis-­induced hypotension in Japan in Japanese healthy adult volunteers, and the for at least 1 hour in patients who have received adequate phase 2 trial (STOP-­AKI) in patients with SA-­AKI29 and fluid resuscitation in accordance with clinical judgement included in an integrated PK analysis. The structural and the recommendations of the surviving sepsis campaign model will contain clearance and volume of distribution guidelines. A minimum of 12 hours without any vasopressor as fixed-­effect parameters. The inter-­patient variability in is needed to consider start of vasopressor therapy as a new the parameter estimates and the random residual error episode. AKI,acute kidney injury; recAP, recombinant human alkaline phosphatase; VP, vasopressor. in the data will be estimated with an appropriate model. Available patient characteristics will be tested as potential covariates affecting PK parameters. Details of the analysis the nominal one-­sided significance levels at interim and will be given in a population PK analysis plan. final analyses for declaring success if data from approxi- mately 700, 850, 1000 and 1400 patients in the main trial Sensitivity analyses and secondary endpoints population will be available. This trial will be considered Sensitivity analyses, based on baseline disease severity a success, and the intervention effective, if the one-­sided score and time from fulfilling inclusion criteria to time p value from the primary efficacy analysis model for the of treatment, will be performed on the primary endpoint. treatment term is lower than the respective nominal one-­ In addition, day 28 all-­cause mortality obtained based on sided significance level. Should the trial be stopped for Kaplan-­Meier curves for time to death up to day 28 will early success at an interim analysis, then the analysis of be compiled separately for the mSOFA categories (≤9 the secondary endpoints within the sequential testing will vs >9) and combined. Finally, a tipping point analysis proceed using the nominal one-­sided significance level in which all ilofotase alfa patients with missing data on as allocated to the primary endpoint at the time of the survival status on day 28 will be considered as being dead, analysis. The predictive probability of success at the main while all placebo patients with missing data will be consid- trial population’s maximum sample size of 1400 patients ered alive, and all possible combinations of missing data will be used to determine if the trial should stop early for between these two extremes will be considered. futility. Futility stopping recommendation in this trial is The most relevant secondary endpoint is Major Adverse considered to be non-­binding. Kidney Events (MAKE) at day 90, defined as dead, or on For this trial design, the operating characteristics (type renal replacement therapy at day 90, or ≥25% decline of I error probability and power) are derived via simulations eGFR on day 28 and day 90 relative to the pre-­AKI reference for a maximum sample size of 1400 patients in the main level. An additional MAKE90a endpoint was added (prior trial population, and the group sequential design. Twenty-­ to unblinding) existing of: death up to and including day eight day all-­cause mortality probability in the placebo 90, >25% decline of eGFR at day 90 relative to pre-­AKI Pickkers P, et al. BMJ Open 2023;13:e065613. doi:10.1136/bmjopen-2022-065613 5 Open access Table 1 Primary and secondary endpoints Objectives Endpoints Primary  To demonstrate an effect of recAP on 28-­day all-­cause mortality. 28-­day all-­cause mortality Secondary  To investigate the effect of recAP on long-­term MAKE MAKE 90: dead by day 90 or on RRT at Day 90 or ≥25% decline in eGFR on both day 28 and day 90 relative to the known or assumed pre-­AKI reference level. Receiving RRT through day 28. Rehospitalisation up to and including day 90.  To investigate the effect of recAP on use of organ support, that is, Days alive and free of organ support through day 28, that is, days alive with no MV, RRT, vasopressors or inotropes MV, RRT, vasopressors or inotropes (with death within 28 days counting as zero days)  To investigate the effect of recAP on LOS in ICU Days alive and out of the ICU through day 28 (with death within 28 days counting as zero days)  To investigate the effect of recAP on 90-­day all-­cause mortality Time to death through day 90 Tertiary/exploratory  To investigate the effect of recAP on 180-­day all-­cause mortality Time to death through day 180  To investigate the effect of recAP on organ function in the first Change in total and individual organ failure scores through day 7 (based on the week mSOFA scores defined as the SOFA score without the GCS component)  To investigate the effect of recAP on short and long-­term renal Days alive and free of RRT through day 28 (with death within 28 days counting as function zero days) MAKE 28: dead by day 28 or on RRT at day 28 or ≥25% decline in eGFR on both day 7/ICU discharge (whichever comes first)  To investigate the effects of recAP on cardiovascular dysfunction Days alive and free of vasopressor and inotropes through day 28 (with death within 28 days counting as zero days)  To investigate the effect of recAP on pulmonary function Days alive and free of MV through day 28 (with death within 28 days counting as zero days)  To investigate the effect of recAP on LOS in hospital and Days alive and out of the hospital through day 90 (with death within 28 days rehospitalisation counting as zero days) Incidence of at least one rehospitalisation at any hospital through day 90  To investigate the effect of recAP on QoL Change in index values, QALY and VAS score based on the EQ-­5D-­5L questionnaire at day 28, day 90 and day 180  To investigate the effects of recAP on urinary excretion of purines The urinary levels of purines (ATP, ADP, AMP, cAMP, and adenosine) through day 4 at selected sites PK  To investigate the PK properties of recAP Population PK Safety  To investigate the safety and tolerability of recAP Generation of anti-­recAP antibodies on day 28 and day 90. Incidence of AEs and SAEs through day 28 The following objectives and endpoints are defined for the main trial population. The same objectives and endpoints will also be assessed and explored in the ‘moderate to severe’ CKD population and the COVID-­19 population without any formal hypothesis testing. cAMP, cyclic AMP; CKD, chronic kidney disease; eGFR, estimated glomerular filtration rate; EQ-­5D-­5L, EuroQoL-­5-­Dimention-­5 Levels; GCS, Glasgow Coma Scale; ICU, Intensive Care Unit; LOS, length of stay; MAKE, major adverse kidney events; mSOFA, modified Sequential Organ Failure Assessment; MV, mechanical ventilation; PK, pharmacokinetics; QALY, quality-­adjusted life years; QoL, quality of life; recAP, recombinant human alkaline phosphatase; RRT, renal replacement therapy; SAEs, serious adverse events; VAS, visual analogue scale. reference level, receiving RRT at day 90 or receiving RRT function, pulmonary function, hospital length of stay and through day 28, rehospitalisation up to and including day quality of life. 90, and an exploratory analysis to evaluate the influence of pre-­existent renal function on the therapeutic efficacy of ilofotase alfa. Additional secondary endpoints are: days ETHICS AND DISSEMINATION alive and free of organ support through day 28 (with This trial will be conducted in accordance with the death within 28 days counting as zero days), days alive protocol and consensus ethical principles derived from and out of the ICU through day 28 (with death within 28 international guidelines, including the Declaration of days counting as zero days) and time to death through Helsinki and Council for International Organisations day 90. of Medical Sciences’ International Ethical Guidelines; Tertiary/explorative endpoints are: time to death applicable International Conference on Harmonisa- through day 180, change in total and individual organ tion GCP Guidelines; and applicable laws and regula- failure scores through day 7 and the effect of recAP on tions. The protocol, substantial protocol amendments, short-­term and long-­term renal function, cardiovascular informed consent form (ICF), investigator’s brochure 6 Pickkers P, et al. BMJ Open 2023;13:e065613. doi:10.1136/bmjopen-2022-065613 Open access (IB) and other relevant documents (eg, any other Author affiliations 1 written information regarding this trial to be provided Intensive Care Medicine, Radboudumc, Nijmegen, The Netherlands 2 Department of Critical Care Medicine, University of Pittsburgh School of Medicine, to the patient or the patient’s legal representative) will Pittsburgh, Pennsylvania, USA be submitted to an IRB/IEC by the investigator and 3 AM-­Pharma BV, Bunnik, The Netherlands reviewed and approved by the IRB/IEC prior to being 4 Department of Critical Care, The University of Melbourne, Melbourne, Victoria, used in the trial. The Standard Protocol Items Recom- Australia 5 mendations for Interventional Trials checklist is added Department of Critical Care, Austin Hospital, Melbourne, Victoria, Australia 6 in online supplemental file 5. Department of Anaesthesiology and Intensive care, Nordsjaellands Hospital, Hillerod, Denmark By the very nature of the trial, it is anticipated that many 7 Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark eligible patients will not be able to give fully informed 8 Berry Consultants, Austin, Texas, USA consent themselves due to various reasons, including 9 Department of Critical Care Medicine, University of Calgary Medical Centre, disease severity, sedation or unconscious state. In this Calgary, Alberta, Canada 10 situation, the patient’s legal representative may provide Intensive Care Department, Universitat Autònoma de Barcelona, Barcelona, Spain 11 written consent, as approved by the institutional-­specific Division of Intensive Care and Emergency Medicine, Department of Internal Medicine, Medical University Innsbruck, Innsbruck, Austria guidelines and legislation in the respective country. 12 ICU and Inserm C1C, University of Limoges, Limoges, France Informed consent may be obtained from an independent 13 Emergency and Critical Care Medicine, The University of Tokyo Hospital, Tokyo, consulting physician or based on an emergency study Japan 14 protocol by the investigator in countries where regula- Department of Critical Care Medicine, University of Pittsburgh, Pittsburgh, tion and institution guidelines permit, and the consent Pennsylvania, USA 15 Department of Critical Care Medicine, Cliniques Universitaires Saint-­Luc, Brussels, procedure has been approved by the institutional review Belgium board (IRB)/independent ethics committee (IEC) or 16 Divisions of Critical Care Medicine and Nephrology, Departments of Anesthesia national authorities, as applicable in specific countries. and Medicine, University of California San Fransisco, San Francisco, California, USA In cases where the initial informed consent is obtained 17 Department of Medicine, University of California, San Diego, California, USA 18 from a legal representative, an independent consulting School of Medicine, University College Dublin, Dublin, Ireland 19 physician, or by the investigator, the patient is asked to Department of Critical Care, King's College London, Guy's & St Thomas' Hospital, London, UK give written informed consent with the most current 20 Division of Intensive Care Medicine, Department of Anesthesiology, Intensive Care version of the ICF(s) as soon as they are able. The master and Pain Medicine, Helsingin Yliopisto Laaketieteellinen tiedekunta, Helsinki, Finland informed consent form is attached as a online supple- 21 PHASTAR, London, UK mental file 6. 22 Intensive Care Unit, Wellington Hospital, Wellington, New Zealand 23 Patients will be assigned a unique patient identification Department of Anesthesiology, Intensive Care and Pain Medicine, Universität number via the Interactive Response Technology (IRT) Münster, Münster, Germany system. Any patient records or datasets that are trans- Contributors PP and ALK co-­drafted the manuscript, participated in the trial ferred to the sponsor will contain this identifier only; protocol design and are accountable for all aspects of the work in ensuring patient names and any information which would make that questions related to the accuracy or integrity of any part of the work are the patient identifiable will not be transferred. All labo- appropriately investigated and resolved. DCA, JA, RB, EvdB, JB, MB, KB, JC, CJD, ratory specimens, evaluation forms, reports and other RF, MJ, BF, KD, JAK, P-­FL, KL, RLM, PTM, MO, VP, SR, PY and AZ all participated in the trial protocol design (as members of the trial steering committee (TSC), records will be identified in a manner designed to main- reviewed and edited the manuscript. All authors read and approved the final version tain patient confidentiality. of the manuscript prior to submission. Irrespective of whether the trial is completed or prema- Funding This work was supported by AM-­Pharma. turely terminated, the sponsor will ensure that the trial Competing interests PP received advisory board consultancy and travel results will be posted on publicly available clinical trial reimbursements from AM-­Pharma. DCA and CJD serve as advisory board registries in accordance with their requirements. In addi- consultants for AM-­Pharma. JA, EvdB and JB are employees of AM-­Pharma. tion, results will be presented at international congresses RB, MB, KD, BF, JAK, P-­FL, VP and PY received advisory board consultancy reimbursements from AM Pharma. KB worked as an employee of Berry Consultants and published in peer-­reviewed journals. KB and acted as a consultant to numerous pharmaceutical and device companies. In addition to answering the question, whether ilofo- KB is currently an employee of and holds stock in AstraZeneca. JC, ALK and tase alfa leads to a reduction in 28-­day all-­cause mortality, SR are consultants to AM-­Pharma. RF received advisory board consultancy the study will provide important information on the effect reimbursements from AM Pharma and additional consulting reimbursements from BioMerieux, Baxter, Pfizer, MSD, Gilead, Shionogi, Grifols and Beckton of ilofotase alfa on MAKE and other clinical outcomes, Dickinson unrelated to the current study. MJ received advisory board consultancy and significantly expand the safety profile of the drug. reimbursements from AM Pharma, additional consulting reimbursement from Baxter Moreover, compared with STOP-­AKI where follow-­up was and Gilead and grant support from Fresenius and Baxter, unrelated to the current limited to 90 days, follow-­up of patients for a period of 180 study. KL received advisory board consultancy reimbursements from AM-­Pharma, owns stock in Amgen and consultant for Biomerieux, Neumora, Seastar and BOA days allows for assessment of potential disease-­modifying Medical. RLM received advisory board consultancy reimbursements from AM effects of ilofotase alfa on kidney function and inves- Pharma and additional consulting reimbursement from BioMerieux, Baxter, Nova tigation of the long-­term effect on mortality and other Biomed, Abiomed, GE Healthcare, Medtronic, Sanofi and Mallinckrodt, unrelated to clinically relevant, patient-­centred and health-­economic the current study. PTM received Trial Steering Committee consultancy payments from AM-­Pharma. Other consultancy payments: FAST Biomedical, Novartis, outcomes. Finally, biomarker analysis may improve the Renibus Therapeutics. MO received speaker honoraria from Fresenius Medical understanding of the mechanism of action of ilofotase Care, Biomerieux, Baxter and Gilead, and research funding from Fresenius Medical alfa. Care, Biomerieux, Baxter and LaJolla Pharma and advisory board consultancy Pickkers P, et al. BMJ Open 2023;13:e065613. doi:10.1136/bmjopen-2022-065613 7 Open access reimbursements from AM-­Pharma. AZ received advisory board consultancy 12 Wen X, Peng Z, Kellum JA. Pathogenesis of acute kidney injury: reimbursements from AM-­Pharma. AZ has received consulting and/or lecture fees effects of remote tissue damage on the kidney. Contrib Nephrol from Astute Medical/BioMerieux, Fresenius, Paion, Guard Therapeutics, and Baxter, 2011;174:129–37. unrelated to the current study. AZ has received grant support from Astute Medical/ 13 Gomez H, Ince C, De Backer D, et al. A unified theory of sepsis-­ induced acute kidney injury: inflammation, microcirculatory BioMerieux, Fresenius and Baxter, unrelated to the current study. dysfunction, bioenergetics, and the tubular cell adaptation to injury. Patient and public involvement Patients and/or the public were not involved in Shock 2014;41:3–11. the design, or conduct, or reporting, or dissemination plans of this research. 14 Verma SK, Molitoris BA. Renal endothelial injury and microvascular dysfunction in acute kidney injury. Semin Nephrol 2015;35:96–107. Patient consent for publication Not applicable. 15 Bagshaw SM, Lapinsky S, Dial S, et al. Acute kidney injury in septic Provenance and peer review Not commissioned; externally peer reviewed. shock: clinical outcomes and impact of duration of hypotension prior to initiation of antimicrobial therapy. Intensive Care Med Supplemental material This content has been supplied by the author(s). It has 2009;35:871–81. not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been 16 Bentala H, Verweij WR, Huizinga-­Van der Vlag A, et al. Removal of peer-­reviewed. Any opinions or recommendations discussed are solely those phosphate from lipid A as a strategy to detoxify lipopolysaccharide. of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and Shock 2002;18:561–6. responsibility arising from any reliance placed on the content. Where the content 17 Koyama I, Matsunaga T, Harada T, et al. Alkaline phosphatases reduce toxicity of lipopolysaccharides in vivo and in vitro through includes any translated material, BMJ does not warrant the accuracy and reliability dephosphorylation. Clin Biochem 2002;35:455–61. of the translations (including but not limited to local regulations, clinical guidelines, 18 Picher M, Burch LH, Hirsh AJ, et al. Ecto 5’-nucleotidase terminology, drug names and drug dosages), and is not responsible for any error and nonspecific alkaline phosphatase. two AMP-­hydrolyzing and/or omissions arising from translation and adaptation or otherwise. ectoenzymes with distinct roles in human airways. 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