Cytokine Adsorption In Patients With Acute-on-chronic Liver Failure .

Sekandarzad et al. Trials(2022) 23:222Clinical Trial Protocol (Continued)that deferral of renal-replacement therapyis mandated. After fulfilling the above inclusion/exclusion criteria, the study teamhas to speak to the ICU and/or nephrology attending physician and ask if he/sheagrees with the statement: “Renal-replacement therapy must be deferred for thispatient.” If the answer is “Yes”, the patientwill be excluded, but could be rescreened for eligibility.When on both questions above it will bereplied “No” by all of the relevant clinicians,the respective patient will be consideredfully eligible for study participation.Study typeInterventionalAllocation: randomized; Intervention model:parallel assignment; Masking: open-labelPrimary purpose: treatment of severe acuteon-chronic liver failurePost-approval/pivotal studyDate of first enrollmentDecember 12, 2021Target sample size51Recruitment statusRecruitment phasePrimary outcome(s)Serum bilirubin reduction after 72 hKey secondary outcomes Survival time (days) from baseline Interleukin-6 after 72 h Liver function parameters (72 h): Quick/INR, AST, ALT, AP, g-GT Blood lactate (72 h) Clinical scores: CLIF-SOFA-score, MELDscore, SOFA score, SAPS II and FIPS score(72 h) Ventilator free days (VeFD) in the first 30days after randomization, where each dayon invasive mechanical ventilation (IMV),non-invasive ventilation (NIV), or ECMO isdefined as ventilator day. VeFD 0, if thepatient dies in the first 30 days afterrandomization Vasopressor free days (VaFD) in the first 30days after randomization, where each daywith any dose of epinephrine,norepinephrine, dobutamine, argipressinor terlipressin is defined as vasopressorday. VaFD 0, if the patient dies in the first30 days after randomization Dialysis free days (DFD) in the first 30 daysafter randomization, where each day onrenal replacement therapy (RRT) is definedas dialysis day. DFD 0 if the patient diesin the first 30 days after randomization A biomarker panel of pro- and antiinflammatory cytokines (blood sampleswill be frozen and stored for later analyses,panel will be determined at the time ofanalysis)1.3. Protocol versionVersion 1.3, 27.01.2022Revision chronology: Version 1.0; submitted to IRB for approval(02.07.2021); revision requested by IRB(05.08.2021) Version 1.1; revised version, consideringprevious request by IRB (09.08.2021); IRBPage 3 of 20Clinical Trial Protocol (Continued)approval (17.08.2021) Version 1.2.; amendment submitted to IRB(04.10.2021); addition of trial registrationinformation, specification ofrandomization tool; IRB approval(19.10.2021)1.4. Trial statusRecruitment phase. Recruiting started December 12, 2021. Estimated trialcompletion by December 31, 2023.1.5. Protocol reporting guidelinesThis clinical trial protocol is following SPIRIT reporting guidelines (seechecklist in the supplement) [1].1.6. FundingThe study is financed by internal funds from the University of FreiburgMedical Center, Department of Medicine III - Intensive Care Medicine. Agrant from the Faculty of Medicine, Freiburg University within thefunding program for clinical trials will be applied for.1.7. Roles and responsibilitiesASe, ASu and DB designed the trial and wrote the first draft of the studyprotocol. EW and EG performed the sample size estimation and set upand wrote the statistical analysis plan. EP advised for specifics related torenal replacement therapy. EP and TW revised the manuscript andadded important content. All co-authors reviewed and approved thefinal version of the manuscript.1.7.1. Sponsor-investigator/Principal InvestigatorDr. Alexander Supady, MPHUniversity of FreiburgMedical CenterDepartment of Medicine III - Intensive Care MedicineHugstetter Str. 5579106 FreiburgGermanyTel.: 49 761 270-73790Fax: 49 761 270-73792E-mail: alexander.supady@uniklinik-freiburg.de1.7.2. Co-Principal InvestigatorPD Dr. Dominik BettingerUniversity of FreiburgMedical CenterDepartment of Medicine IIHugstetter Str. 5579106 FreiburgGermanyTel.: 49 761 270-34010 or -32440E-mail: dominik.bettinger@uniklinik-freiburg.de1.7.3. Co-Principal InvestigatorDr. Asieb SekandarzadUniversity of FreiburgMedical CenterDepartment of Medicine III - Intensive Care MedicineHugstetter Str. 5579106 FreiburgGermanyTel.: 49 761 270-33322E-mail: asieb.sekandarzad@uniklinik-freiburg.de1.7.4. Cooperating investigatorDr. Eric Peter PragerUniversity of FreiburgMedical CenterDepartment of Medicine IVHugstetter Str. 5579106 FreiburgGermanyTel.: 49 761 270-34140E-mail: eric.peter.prager@uniklinik-freiburg.de1.7.5. Trial statisticianDr. Erika GrafUniversity of Freiburg

Sekandarzad et al. Trials(2022) 23:222Clinical Trial Protocol (Continued)Institute of Medical Biometry and Statistics (IMBI)Stefan-Meier-Straße 2679104 FreiburgGermanyTel.: 49 761 270-83743E-mail: erika.graf@uniklinik-freiburg.de1.7.6. Role of study sponsor and fundersThe study will be financed from internal funds from the University ofFreiburg, Medical Center, Department of Medicine III - Intensive CareMedicine and the University of Freiburg Faculty of Medicine. Thesponsor-investigator/principal investigator is an employee of Universityof Freiburg Medical Center. The sponsor-investigator/principal investigator is independently responsible for collection, management, analysis,and interpretation of data, writing of the report(s) and the decision tosubmit the report for publication.IntroductionBackground and rationaleSystemic inflammation in decompensated liver cirrhosis andacute-on-chronic liver failure (ACLF)Liver cirrhosis is a major healthcare problem. Amongaffected patients mortality is high. In Germany, in 2015more than 13,000 patients died owing to decompensatedcirrhosis [2]. The clinical course of cirrhosis can beseparated in compensated and decompensated cirrhosis.Patients with compensated cirrhosis are largelyasymptomatic and the development of decompensatingevents is a major hallmark in the course of the disease asmedian survival decreases from 12 years to less than 2years [3]. The most important decompensating eventsare the development of variceal bleeding, ascites, hepaticencephalopathy, and bacterial infections [4]. Previously,liver cirrhosis has been described as a systemic diseaseaffecting almost any other organ system [4, 5]. Thedevelopment of extrahepatic organ complications indecompensated cirrhosis has been identified as a majorprognostic milestone and has been described as acuteon-chronic liver failure (ACLF) [6]. ACLF is understoodas a dynamic process and may evolve within days leadingto multi-organ failure with renal failure being the mostcommon organ involvement (56%), followed by liver andcoagulation failure (44% and 28%, respectively) [6, 7].ACLF is associated with a high 28-day mortality rangingfrom 22% to 77% in patients with multi-organ failure [6].During recent years, systemic inflammation has beenrecognized as a major driver of hepatic decompensationand progression of liver cirrhosis to ACLF. Systemicinflammation steadily increases with the progression ofdecompensated liver cirrhosis and the development ofACLF [4, 8]. Importantly, systemic inflammation wasdescribed as an important trigger for the development ofextrahepatic organ failures, such as renal failure,development of hepatopulmonary syndrome, cirrhoticcardiomyopathy, and hepatic encephalopathy [4, 9, 10].The evolution of systemic inflammation in patients withPage 4 of 20progressing hepatic decompensation and ACLF is due toan increase in bacterial translocation in the intestine [4,11]. Bacterial translocation is associated with portalhypertension which is one of the major drivers ofdecompensation in cirrhotic patients and determines ahallmark in the course of disease [4, 12]. Damageassociated molecular patterns (DAMPs) and pathogenassociated molecular patterns (PAMPs) may serve as asurrogate for bacterial translocation in patients with livercirrhosis [4]. In summary, systemic inflammation is particularly relevant in the pathogenesis of acute hepatic decompensation and is also associated with reducedsurvival [13]. Therefore, elimination of PAMPs, DAMPs,and inflammatory cytokines in addition to establishedtherapeutic approaches aiming at a reduction of bacterialtranslocation and mitigation of portal hypertension mayhelp control excessive inflammatory activity and thussupport hepatic recompensation. This may ultimatelylead to improved survival.Previous in-vitro examinations and studies in noncirrhotic inflammatory disorders have shown that proinflammatory cytokines, DAMPs, and PAMPs can effectively be removed by extracorporeal hemoadsorption inthe CytoSorb adsorber [14].CytoSorb cytokine adsorberThe CytoSorb adsorber (CytoSorbents Corporation,Monmouth Junction, NJ, USA) is a medical device approvedfor the removal of cytokines, bilirubin, and myoglobin byhemoadsorption. The adsorber can be installed in any kindof extracorporeal blood circuit, such as extracorporealmembrane oxygenation (ECMO) or continuous renalreplacement therapy (CRRT), via an associated tubing andconnector system using Luer-Lock connections.The adsorber consists of a cylindrical cartridge filled withtiny, highly porous, biocompatible, and ene-divinyl-benzenepolymer beads with a total surface area of 40,000 m2 peradsorber [15]. The polymer adsorbs hydrophobic moleculeswithin the 5–55 kDa range; these include, among others, bilirubin, and vasoactive cytokines, associated with acute decompensation in ACLF [4, 15]. Within the device, molecules areadsorbed based on their physicochemical properties (molecular weight, size, solvation tendency) independent of physiological or pathophysiological functions. Therefore, not onlyharmful substances, but also pathophysiologically “necessary”or “desired” substances, e.g., anti-inflammatory cytokines ordrugs, may be adsorbed. The adsorption capacity is postulatedto be concentration-dependent, so that low physiologicallevels are not significantly affected [16].Rationale for the study designThe CYTOHEP study is designed as a prospective,randomized, single-center, open-label, controlled

Sekandarzad et al. Trials(2022) 23:222intervention trial to assess the benefit of extracorporealhemoadsorption using the CytoSorb device in patientswith acute-on-chronic liver failure. The primary goal forthis trial is to assess whether the CytoSorb device usedin addition to CRRT will be able to significantly reducebilirubin in the patient blood as compared to the controlgroup treated with CRRT alone (i.e., without extracorporeal hemoadsorption).Within this trial, CRRT will be initiated early, i.e., inpatients with acute kidney injury (AKI) Kidney Disease:Improving Global Outcome (KDIGO) stage 3 (see the“Subject inclusion criteria,” “Subject exclusion criteria,”and “Description of study plan and interventions”section for details). For safety assessment, a third groupwill be assessed without early initiation of CRRT andextracorporeal hemoadsorption. After trial inclusion, allpatients will be randomized in a 1:1:1 fashion in one ofthe study groups.The rationale for this study is based on considerationsabout the role of systemic inflammation in acutedecompensation of liver cirrhosis and ACLF (see the“Specific objectives” section), in-vitro data comparingthe effectiveness of MARS (molecule adsorbent recirculating system) and CytoSorb for the removal of molecules with a pathophysiological role in acute-on-chronicliver failure, and recent reports on the successful use ofextracorporeal hemoadsorption in combination withCRRT in critically ill patients with acute liver dysfunction [17–21]. The choice of the primary endpoint bilirubin reduction after 72 h is based on results from thesereports describing the successful elimination of bilirubinwith CytoSorb. Additionally, a broad array of clinicaland inflammatory parameters will be assessed to betterunderstand the mode of action and molecular effects ofthe CytoSorb adsorber. The trial is planned to befollowed by a subsequent multi-center trial.Risks and benefitsPotential risks and benefits associated with the studyprocedures Besides study-specific interventions as outlined in this protocol, patients in all study groups, CRRTwith CytoSorb, CRRT without CytoSorb, and no CRRT,will be treated according to establish standard of care(SOC), and current guidelines, treatment standards, andrecommendations will apply. In addition to SOC treatment, patients in both the CRRT with the CytoSorb groupand the CRRT without the CytoSorb group will be treatedwith continuous renal replacement therapy (CRRT). Patients in the CRRT with CytoSorb group will receiveextracorporeal hemoadsorption for a total duration of 72h. The adsorber will be included in the CRRT machine.For patients in the no CRRT group the use of CRRT willbe delayed until CRRT will be otherwise indicated by thePage 5 of 20treating physicians or one or more of the following criteriawill evolve: a serum potassium level of 6.0 mmol or moreper liter, a pH of 7.20 or less due to metabolic acidosis ora serum bicarbonate level of 12 mmol per liter or less, evidence of severe respiratory failure based on a ratio of thepartial pressure of arterial oxygen to the fraction of inspired oxygen of 200 or less and clinical perception of volume overload.Within this trial, both in the CRRT with CytoSorbgroup and in the CRRT without CytoSorb group CRRTwill be initiated in patients with ACLF and AKI KDIGOstage 3. The optimal timing of initiation of CRRT inpatients with acute kidney injury (AKI) is a matter ofdebate. Generally, a rather restrictive strategy forinitiation of hemodialysis is recommended, i.e., initiatingCRRT when life-threatening deterioration in fluid, electrolyte, or acid-base balance evolves that cannot be managed by conservative treatment [22]. Several randomizedcontrolled trials (RCTs) and meta-analyses have compared early versus delayed initiation strategies for CRRTwith, in part, conflicting results. One single-center RCTshowed a mortality benefit (90 days) for early initiationof CRRT in critically ill patients, i.e., initiation of CRRTwhen reaching AKI KDIGO stage 2 compared to initiation of CRRT not before reaching AKI KDIGO stage 3(39.3% vs. 54.7%, p 0.03) [23]. A subsequent multicenterRCT, however, did not show a difference in mortality at60 days when comparing initiation of CRRT in AKIKDIGO stage 3 to an even more restrictive strategy(48.5% vs. 49.7%), i.e., initiation only when potentiallylife-threatening complications (severe hyperkalemia,metabolic acidosis, pulmonary edema) occurred or ablood urea nitrogen (BUN) level higher than 112 mg perdeciliter or oliguria for more than 72 h were present. Inthis trial, adverse events were similar in both groups, besides more frequent occurrence hypophosphatemia andcatheter-related blood stream infections in the earlyinitiation group (hypophosphatemia: 22% vs. 15%, p 0.03, infections: 10% vs. 5%, p 0.03) [24].Recently, another multicenter RCT did not reveal adifference in mortality at day 90 (43.9% vs. 43.7%)when comparing an early initiation strategy of CRRT(i.e., within 12 h after reaching AKI KDIGO stage 2or 3) with a delayed initiation strategy (i.e., when lifethreatening complications of AKI occurred, or AKIstage 2 or 3 persisted for more than 72 h). However,adverse events occurred more frequently in the earlyinitiation group (23% vs. 16.5%, p 0.001) and morepatients in this group remained dependent on CRRTat 90 days (10.4% vs. 6%, relative risk: 1.74; 95% CI,1.24 to 2.43) [25].In a meta-analysis of nine studies comparing early vs.late CRRT initiation strategies in 1879 patients, no difference in survival at 28 days was found (44% vs. 43%) [26].

Sekandarzad et al. Trials(2022) 23:222Finally, another RCT showed that further delayingCRRT in patients with oliguria for more than 72 h orBUN 112 mg/dl even without life-threatening complications was not beneficial but even associated with anincreased 60-day mortality risk (HR 1.65; 95% CI 1.09–2.50, p 0.018) [27].So far, the above-described strategies for initiation ofCRRT have not been assessed in patients with ACLFand the results from these trials cannot easily be extrapolated to the patient cohort eligible for this trial. Therefore, in patients eligible for the CYTOHEP study, theoptimal timing for initiation of CRRT remains unclear,so far [28, 29].At least some of the patients eligible for studyparticipation in the CYTOHEP trial will suffer fromcoagulation disorders due to liver function failure andstandard anticoagulation strategy for CRRT withunfractionated heparin can be difficult. On the otherhand, citrate anticoagulation, that is usually used inpatients with coagulation abnormalities, can rarely causemetabolic acidosis in patients with liver disease as citratecan accumulate in patients with severe hepatic metabolicabnormalities.Considering the above summarized evidence, weconsider initiation of CRRT in patients with AKIKDIGO stage 3 within this trial reasonable and safe. Inorder to be able to thoroughly assess the safety of thestudy interventions within this trial, a third groupwithout CRRT will be assessed.In addition to routine diagnostics, additional bloodsamples are necessary within the scope of the study at 0h/baseline/screening and 72 h (43.4 ml each). In theCRRT with CytoSorb group and the CRRT withoutCytoSorb group, the blood samples are taken via anarterial catheter (A. radialis or A. brachialis) or a centralvenous catheter, which are necessary for regular therapymonitoring, drug treatment, and renal replacementtherapy—no additional vascular punctures are required.Not all patients in the no CRRT group will require anarterial catheter or a central venous catheter. In thesepatients, blood draws are scheduled to be combined withroutine blood draws necessary for treatment guidance, sothat no additional vascular punctures are required.Potential risks and benefits associated with theCytoSorb device A potential concern with the use ofthe CytoSorb device in patients eligible for this study isthe adsorption of drugs, most specifically antibioticsrequired for the treatment of the patients with ACLF.According to current data and knowledge, penicillins,including combination drugs ampicillin/sulbactam orpiperacillin/tazobactam, or cephalosporines, which couldbe required in this patient population in the case ofbacterial infection, are not removed from the patient’sPage 6 of 20blood to a clinically relevant extent [30, 31]. If theadministration of a drug within the adsorption spectrumof the CytoSorb adsorber is necessary for a patient inthe CRRT with CytoSorb group and no equivalentalternative is available, the therapy with the CytoSorbadsorber can be terminated at any time.In a randomized controlled pilot trial in 34 COVID-19patients on ECMO (CYCOV study), an association between cytokine adsorption and increased mortality hasbeen described [32]. In contrast to these observations, invarious retrospective analyses, the use of CytoSorb was described as safe and positive effects have been reported[33–37]. Based on the above-described inflammationhypothesis as a driver of acute decompensation in livercirrhosis and the observed association between inflammation and mortality in these patients, we postulate a beneficial effect of cytokine adsorption by mitigation ofincreased inflammatory processes and cascades. However,respecting the results from the CYCOV study, we willcontinuously monitor and assess the safety of the intervention and closely monitor clinical courses and mortalityin all study groups (see also the “Monitoring” and “Earlytermination of the study” section).Patients eligible for this study have limited therapeuticoptions and mortality is high. Based on pathophysiologicalconsiderations and preliminary experience using theCytoSorb device in these patients we postulate a positivebenefit-risk ratio for the intervention.Study aim and choice of comparatorThe aim of the CYTOHEP study is to investigate theinfluence of cytokine adsorption with the CytoSorbadsorber in combination with CRRT on serum bilirubinconcentrations, humoral inflammation parameters, liverfunction parameters, and patient survival undercontrolled conditions in patients with ACLF. Since thereis equipoise for the use of extracorporeal cytokineadsorption with CRRT in these patients, we directlycompare three different treatment strategies, CRRT withCytoSorb, CRRT without CytoSorb, and no CRRT,under otherwise equal conditions and treatmentstandards and regimens.Extracorporeal hemoadsorption with the CytoSorbdevice has to be incorporated into an extracorporealblood circuit, such as a CRRT circuit. As describedabove (“Potential risks and benefits associated with thestudy procedures” section), the evidence as to when toinitiate CRRT in the patients eligible for this study (seethe “Subject inclusion criteria,” “Subject exclusioncriteria” sections for inclusion and exclusion criteria) isincomplete, and therefore, the optimal timing forinitiation of CRRT remains unclear, so far. Foradditional safety within this trial in order to be able todetect early warning signs of a negative effect of early

Sekandarzad et al. Trials(2022) 23:222CRRT or CytoSorb compared to a delayed strategy ofinitiation of CRRT, we included a third study group, theno CRRT group. In this group, patients will be treatedwithout CRRT and without CytoSorb. If indicated,CRRT will be initiated late and only when lifethreatening complications occur (see the “Potential risksand benefits associated with the study procedures” section). This approach has been tested in a recent RCTand was not inferior to the approach for early initiationof CRRT as applied in the CRRT with CytoSorb andCRRT without CytoSorb groups [25].ObjectivesSpecific objectivesThe aim of this study is to show under controlled andrandom

Background: Liver cirrhosis is a major healthcare problem and the mortality rate is high. During recent years, systemic inflammation has been recognized as a major driver of hepatic decompensation and progression of liver cirrhosis to acute-on-chronic liver failure (ACLF). The aim of the CYTOHEP study is to assess the impact of extracorporeal