Special Populations

The Journal of Clinical Pharmacology Pharmacokinetics of Serelaxin in Patients With 2016, 56(4) 474–483 © 2015 The Authors. The Journal of Severe Renal Impairment or End-Stage Renal Clinical Pharmacology Published by Wiley Periodicals, Inc. on behalf of American College of Clinical Disease Requiring Hemodialysis: A Single-Dose, Pharmacology Open-Label, Parallel-Group Study DOI: 10.1002/jcph.607

Marion Dahlke, MD, PhD1, Atef Halabi, MD, PhD2, Jasna Canadi, PhD1, Chiaki Tsubouchi, PhD1, Surendra Machineni, MSc3, and Yinuo Pang, PhD4

Abstract Serelaxin, a recombinant human relaxin-2 hormone, is in clinical development for treating acute heart failure. This open-label, parallel-group study investigated serelaxin pharmacokinetics (PK) after a single 4-hour intravenous infusion (10 mg/kg) in patients with severe renal impairment (n 6) or ¼ end-stage renal disease (ESRD) requiring hemodialysis (PK on the day of dialysis [n 6] or during dialysis-free interval [n 6]), compared with ¼ ¼ matched healthy subjects (n 18). In all participants, serum serelaxin concentration peaked at the end of infusion and subsequently declined with mean ¼ terminal elimination half-life of 6.5–8.8 hours. Compared with healthy subjects, a moderate decrease in serelaxin systemic clearance (37%–52%) and increase in its exposure (30%–115%) were observed in all patients. During the 4-hour hemodialysis in ESRD patients, 30% serelaxin was removed, with hemodialysis clearance constituting approximately 52% of total systemic clearance. Serelaxin was well tolerated with no deaths, serious adverse events (AE), or AE-related discontinuations. Antiserelaxin antibodies were not detected in any participant. Given the shallow dose-response relationship observed with serelaxin in clinical studies and its wide therapeutic window, the observed PK differences in patients with severe renal impairment compared with healthy subjects are unlikely to pose a safety risk and do not warrant a predefined dosage adjustment in such patients.

Keywords serelaxin, pharmacokinetics, renal impairment, end-stage renal disease

Serelaxin, currently in clinical development for the treatment of acute heart failure (AHF), is a recombinant peptide identical in structure to human relaxin-2, a naturally occurring hormone that is believed to mediate hemodynamic and vascular changes during pregnancy.1 Serelaxin stimulates the vascular signaling pathways with Abbreviations: AE, adverse events; AHF, acute heart failure; AUC, short- and long-term effects on hemodynamics.1,2 Data area under the serum concentration-time curve; BMI, body mass fi are available on the efficacy and safety of serelaxin in index; Cmax, maximum serum concentration; CI, con dence interval; CV, cardiovascular; DBP, diastolic blood pressure; eGFR, estimated patients with AHF from 2 randomized, double-blind, glomerular filtration rate; ELISA, enzyme-linked immunosorbent placebo-controlled, parallel-group, multinational clinical assay; ESRD, end-stage renal disease; GMR, geometric mean ratios; trials—the phase 2 Pre-RELAX-AHF and the pivotal HF, heart failure; IV, intravenous; PK, pharmacokinetics; PR, pulse phase 3 RELAX-AHF.3,4 In these studies, a 48-hour rate; RELAX-AHF, RELAXin in Acute Heart Failure; SAE, serious intravenous (IV) infusion of serelaxin, 30 mg/kg/day adverse event; SBP, systolic blood pressure; SD, standard deviation. significantly improved dyspnea and was well tolerated 1Novartis Pharma AG, Basel, Switzerland with favorable effects on long-term clinical outcomes.3,4 In 2Clinical Research Services GmbH, Kiel, Germany 3 addition, the RELAX-AHF trial demonstrated that ser- Novartis Healthcare Pvt Ltd, Hyderabad, India 4Novartis Institutes for BioMedical Research, Cambridge, MA, USA elaxin, along with standard therapy, significantly reduced the incidences of in-hospital worsening heart failure (WHF) This is an open access article under the terms of the Creative through day 5 and all-cause and cardiovascular (CV) Commons Attribution-NonCommercial License, which permits use, mortality through day 180; however, no significant effects distribution and reproduction in any medium, provided the original were observed for days alive and out of hospital and CV work is properly cited and is not used for commercial purposes. death or readmission for heart failure (HF) or renal failure Submitted for publication 11 June 2015; accepted 31 July 2015. up to day 60.3 An ongoing phase 3 trial, RELAX-AHF-2, fi Corresponding Author will con rm the effects of serelaxin on reducing CV death Yinuo Pang, PhD, Novartis Institutes for BioMedical Research, 200 over 180 days after treatment and in-hospital WHF through Technology Square, Cambridge, MA 02139, USA day 5 (primary endpoints), and to assess several important Email: [email protected] 2Dahlke et al. The Journal of Clinical Pharmacology / Vol XX No XX 20154753

clinicalcreatinine outcomes clearance (secondary levels calculated endpoints) according in a large patient to the toused assess were the Fresenius safety, 4008s tolerability, (Dialysator and immunogenicity FX80, surface area of populationsimplified Modi of approximatelyfication of Diet 6800 in Renal patients Disease with formula AHF serelaxin1.8 m2), Fresenius in all the participants.5008s (Dialysator FX80, surface area (ClinicalTrials.gov(15 mL/[min 1.73 midenti2] fierestimated NCT01870778). glomerular filtration 1.8 m2), Fresenius 5008s (Dialysator FX1000, surface ratePatients [eGFR] with<� 30 mL/[min AHF� are1.73 often m2 diagnosed]). Patients inwith groups renal 2 area 2.2 m2), and Gambro AK 200 ultra (Dialysator insufandfi 3ciency were or included experience if� worseningthey had ofESRD renal requiring function MethodsPolyflux P210H). For ESRD patients with PK assess- duringhemodialysis. hospitalization. The vital A signs meta-analysis for patients of in groupsHF studies 1 to Studyments Design performed and Participants during the dialysis-free interval, showed3, at screening that 63% and baseline of patients visits, had were some required degree to ofhave renal the Theserelaxin study infusion protocol was was started reviewed after an and interval approved of at least by dysfunction,following: systolic with 29% blood having pressure moderate (SBP) ranging to severe from renal 110 the12 hours Research following Ethics the Committee previous hemodialysis at Ethikkommission with PK dysfunction.to 170 mm Hg,5 An diastolic analysis blood of the pressure Acute Decompensated (DBP) ranging Schleswig-Holstein,sample collection and Germany. safety/tolerability The study was assessments designed, Heartfrom 60 Failure to 105 National mm Hg, andRegistry pulse (ADHERE) rate (PR) between data base 45 and of conducted,being performed and reported over the in following accordance 48 with hours the (until ICH 118,465100 bpm. patients The corresponding hospitalized inclusionwith AHF criteria showed for that healthy 91% Harmonizedday 3) and prior Tripartite to the Guidelines subsequent for hemodialysis. Good Clinical All ofsubjects patients were had eGFR some degree90 mL/(min of renal dysfunction,1.73 m2); matching of whom in Practice,participants the were applicable required local to remain regulations, at the study and site the 64%terms had of race, at least age a ( � moderate10 years), renal sex,� anddysfunction. BMI ( 615%)In the to Declarationfor approximately of Helsinki. 3 days All to facilitate participants baseline, provided PK, Pre-RELAX-AHFan individual patient and� with RELAX-AHF renal impairment trials, in the groups� analyses 1, 2, writtenand safety/tolerability informed consent assessments, before any with study an procedures additional ofor 3; serelaxin SBP ranging systemic from clearance 100 to 150 in mm patients Hg; DBP with ranging renal wereambulatory conducted. end-of-study visit on day 15. dysfunctionfrom 60 to 95 indicated mm Hg; andno clinically PR between relevant 50 and impact 100 bpm. of This was a phase 1, open-label, parallel-group study impairedThe keyrenal function exclusion on thecriteria pharmacokinetics were as follows: (PK) of conductedPharmacokinetic in patients Assessments with severe and renal Analyses impairment and serelaxin.(1) clinically However, significant these electrocardiogram were mostly patients abnormalities; with mild thoseSerum with samples ESRD for PKrequiring analysis hemodialysis. were collected The during control the to(2) moderate history of renal hypersensitivity dysfunction to and the a study relatively drug small or to grouptreatment comprised and follow-up demographically periods (time matched points: pretreat- healthy numberdrugs of of a patients similar withclass; severe (3) use renal of impairmentother investigational (n 12). subjectsment, 15 with minutes, normal and renal 1, 2, 3,function. 4, 4.25, As 5, 6, shown 7, 8, 9, in 10, Figure 11, 12, 1, Indrugs addition, at the time the available of study enrollment; serelaxin PK (4) data presence in these¼ of any 12 the16, 24, study 28, participants 36, and 48 hours were after grouped the start according of infusion) to their and patientsnoncontrolled were limitedand clinically to the signi steady-stateficant disease, serum surgical, concen- renalon day function 15 (end status of study). as follows: Specifi patientscally for with group severe 2 patients, renal trationsor medical at condition 12 hours that during could infusion have affected (data the on studyfile). impairmentwho had hemodialysis (group 1), performed patients from with 6 ESRD to 10 hours requiring post– Therefore,outcome or itthat was would deemed have placed important the patient to systematically at an undue hemodialysisserelaxin administration with PK assessments (4-hour dialysis on the period),day of dialysis serum assessrisk, as the judged effects by of the severe investigator; renal dysfunction and (5) baseline and renal and (groupsamples 2), for and PK patients analyses with were ESRD collected requiring from hemodialysis both the line failureother laboratory on the PK parameters of serelaxin. at screening This aspect outside has accept- been withentering PK (LINE assessments IN) and during that exitingthe dialysis-free (LINE OUT) interval the addressedable limits in asthe judged present study by the (ClinicalTrials.gov investigator. In identiaddition,fier (groupdialyzer. 3). In Healthy addition, subjects hemodialysis (group 4)fluid were samples matched were to NCT01875523),patients with severe which renal was conducted impairment/ESRD in a selected were individualcollected from patients these in patients groups 16 tohours 3. prior to the start of populationexcluded from of patients the study with if severe they had renal hemoglobin impairment levels and dialysisMale and or as female fractions participants during the aged 4-hour between dialysis 18 period and those<9.0 g/dL with at end-stage screening renal and received disease treatment (ESRD) requiring with any 75for years, assesment with of body serelaxin weight concentrations.50 kg, body The mass dialysate index hemodialysis.cytostatic drug The or nitrate. primary Healthy objective subjects of this were study excluded was to (BMI)was collected of 18 to in 3520-L kg/m bottles,2, and with� an 20-mL ability aliquots to communicate (or 0.1% evaluateif they had the used PK any of serelaxin prescription after drugs a single within 4-hour 4 weeks IV wellof the with respective the investigator fraction) being were taken included from in each the bottle,study. infusionprior to of the 10 mg/kg initial serelaxin dosing in and/orpatients withover-the-counter severe renal Inpooled, addition, and stored for patients at 3°C to in5°C group for the 1, duration inclusion of dialysis. in the impairmentmedications or within ESRD 2 requiring weeks prior hemodialysis to the initial in compari- dosing, studyAt the required end of evidence the procedure, of severe 3-mL renal samples impairment of the as sonor had with tested healthy positive subjects. for hepatitisThe secondary B or C. objectives were indicatedthoroughly by mixed clinically pooled signi dialysateficant abnormal aliquots creatinine were frozen and Patients in groups 1 to 3 were enrolled in parallel into at 70°C and later shipped on dry ice for analysis. the study. Healthy subjects were matched pairwise and �The serum serelaxin concentrations were determined enrolled after the corresponding patient with renal by a validated, commercially available enzyme-linked impairment/ESRD had completed serelaxin treatment immunosorbent assay (ELISA) kit (R&D Systems, and PK blood sample collection. Minneapolis, Minnesota). Briefly, the ELISA method Study participants received a single open-label, 4-hour used a monoclonal antibody specific for relaxin-2 as the IV infusion of 10 mg/kg serelaxin on day 1. This dosing capture reagent and an enzyme-linked polyclonal anti- regimen (instead of 30 mg/kg/day for 48 hours that body specific for relaxin-2 as the detection reagent. The was implemented in the serelaxin clinical development lower limit of quantification was 15.6 pg/mL. The same studies, including the ongoing phase 3 RELAX-AHF2 kit was used to determine the serelaxin concentration in study) was chosen due to the feasibility challenges with the dialysate for group 2, following validation, to ensure regard to the timing of PK assessments and the no matrix interference to the assay. The lower limit of intermittent hemodialysis (every other day) and for safety quantification in the dialysate was 31.3 pg/mL. considerations. A short infusion was selected over an IV Noncompartmental PK analysis was performed using bolus to minimize the potential risks associated with a WinNonlin Phoenix (Version 6.2). The evaluated PK high initial serum concentration following an IV bolus. parameters are described in Table 1. The key PK For patients with ESRD undergoing PK assessments on parameters to compare serelaxin exposure between the the day of hemodialysis, the dialysis procedure was patient groups and matched healthy subjects were area started 2 hours ( 15 minutes) after the end of infusion and under the serum concentration-time curve (AUC) from � Figurelasted1. forStudy approximately design—a phase 4 hours. 1, open-label, The dialysis parallel-group machines study. time 0 to infinity (AUC ) and the observed maximum 1 476Dahlke et al. The Journal of Clinical Pharmacology / Vol 56 No 4 20163 creatinine clearance levels calculated according to the used were Fresenius 4008s (Dialysator FX80, surface area simplified Modification of Diet in Renal Disease formula 1.8 m2), Fresenius 5008s (Dialysator FX80, surface area (15 mL/[min 1.73 m2] estimated glomerular filtration 1.8 m2), Fresenius 5008s (Dialysator FX1000, surface rate [eGFR] <� 30 mL/[min� 1.73 m2]). Patients in groups 2 area 2.2 m2), and Gambro AK 200 ultra (Dialysator and 3 were included if� they had ESRD requiring Polyflux P210H). For ESRD patients with PK assess- hemodialysis. The vital signs for patients in groups 1 to ments performed during the dialysis-free interval, 3, at screening and baseline visits, were required to have the serelaxin infusion was started after an interval of at least following: systolic blood pressure (SBP) ranging from 110 12 hours following the previous hemodialysis with PK to 170 mm Hg, diastolic blood pressure (DBP) ranging sample collection and safety/tolerability assessments from 60 to 105 mm Hg, and pulse rate (PR) between 45 and being performed over the following 48 hours (until 100 bpm. The corresponding inclusion criteria for healthy day 3) and prior to the subsequent hemodialysis. All subjects were eGFR 90 mL/(min 1.73 m2); matching in participants were required to remain at the study site terms of race, age ( �10 years), sex,� and BMI ( 15%) to for approximately 3 days to facilitate baseline, PK, an individual patient� with renal impairment in groups� 1, 2, and safety/tolerability assessments, with an additional or 3; SBP ranging from 100 to 150 mm Hg; DBP ranging ambulatory end-of-study visit on day 15. from 60 to 95 mm Hg; and PR between 50 and 100 bpm. The key exclusion criteria were as follows: Pharmacokinetic Assessments and Analyses (1) clinically significant electrocardiogram abnormalities; Serum samples for PK analysis were collected during the (2) history of hypersensitivity to the study drug or to treatment and follow-up periods (time points: pretreat- drugs of a similar class; (3) use of other investigational ment, 15 minutes, and 1, 2, 3, 4, 4.25, 5, 6, 7, 8, 9, 10, 11, 12, drugs at the time of study enrollment; (4) presence of any 16, 24, 28, 36, and 48 hours after the start of infusion) and noncontrolled and clinically significant disease, surgical, on day 15 (end of study). Specifically for group 2 patients, or medical condition that could have affected the study who had hemodialysis performed from 6 to 10 hours post– outcome or that would have placed the patient at an undue serelaxin administration (4-hour dialysis period), serum risk, as judged by the investigator; and (5) baseline and samples for PK analyses were collected from both the line other laboratory parameters at screening outside accept- entering (LINE IN) and that exiting (LINE OUT) the able limits as judged by the investigator. In addition, dialyzer. In addition, hemodialysis fluid samples were patients with severe renal impairment/ESRD were collected from these patients 6 hours prior to the start of excluded from the study if they had hemoglobin levels dialysis and as fractions during the 4-hour dialysis period <9.0 g/dL at screening and received treatment with any for assesment of serelaxin concentrations. The dialysate cytostatic drug or nitrate. Healthy subjects were excluded was collected in 20-L bottles, with 20-mL aliquots (or 0.1% if they had used any prescription drugs within 4 weeks of the respective fraction) being taken from each bottle, prior to the initial dosing and/or over-the-counter pooled, and stored at 3°C to 5°C for the duration of dialysis. medications within 2 weeks prior to the initial dosing, At the end of the procedure, 3-mL samples of the or had tested positive for hepatitis B or C. thoroughly mixed pooled dialysate aliquots were frozen Patients in groups 1 to 3 were enrolled in parallel into at 70°C and later shipped on dry ice for analysis. the study. Healthy subjects were matched pairwise and �The serum serelaxin concentrations were determined enrolled after the corresponding patient with renal by a validated, commercially available enzyme-linked impairment/ESRD had completed serelaxin treatment immunosorbent assay (ELISA) kit (R&D Systems, and PK blood sample collection. Minneapolis, Minnesota). Briefly, the ELISA method Study participants received a single open-label, 4-hour used a monoclonal antibody specific for relaxin-2 as the IV infusion of 10 mg/kg serelaxin on day 1. This dosing capture reagent and an enzyme-linked polyclonal anti- regimen (instead of 30 mg/kg/day for 48 hours that body specific for relaxin-2 as the detection reagent. The was implemented in the serelaxin clinical development lower limit of quantification was 15.6 pg/mL. The same studies, including the ongoing phase 3 RELAX-AHF2 kit was used to determine the serelaxin concentration in study) was chosen due to the feasibility challenges with the dialysate for group 2, following validation, to ensure regard to the timing of PK assessments and the no matrix interference to the assay. The lower limit of intermittent hemodialysis (every other day) and for safety quantification in the dialysate was 31.3 pg/mL. considerations. A short infusion was selected over an IV Noncompartmental PK analysis was performed using bolus to minimize the potential risks associated with a WinNonlin Phoenix (Version 6.2). The evaluated PK high initial serum concentration following an IV bolus. parameters are described in Table 1. The key PK For patients with ESRD undergoing PK assessments on parameters to compare serelaxin exposure between the the day of hemodialysis, the dialysis procedure was patient groups and matched healthy subjects were area started 2 hours ( 15 minutes) after the end of infusion and under the serum concentration-time curve (AUC) from lasted for approximately� 4 hours. The dialysis machines time 0 to infinity (AUC ) and the observed maximum 1 4Dahlke et al. The Journal of Clinical Pharmacology / Vol XX No XX 20154773

Tablecreatinine 1. Pharmacokinetic clearance levels Parameters calculated Assessed according to the used were Fresenius 4008s (Dialysator FX80, surface area simplified Modification of Diet in Renal Disease formula 1.8 m2), Fresenius 5008s (Dialysator FX80, surface area Parameters Description (15 mL/[min 1.73 m2] estimated glomerular filtration 1.8 m2), Fresenius 5008s (Dialysator FX1000, surface AUC � The area under� the serum2 concentration-time curve from time 0 to infinity2 (mass time/volume) rate1 [eGFR] <30 mL/[min 1.73 m ]). Patients in groups 2 area 2.2 m ), and� Gambro AK 200 ultra (Dialysator AUCandt_in 3 were The included area under if� the they serum had concentration-time ESRD requiring curve during thePoly dialysisflux interval P210H). based For on samples ESRD collected patients at the with entry PKof the assess- dialyzer AUChemodialysis.t_out The The area vital under signs the serum for patients concentration-time in groups curve 1 to during thements dialysis performedinterval based on during samples collected the dialysis-free at the exit of dialyzer interval, CD The concentration of the drug in the pooled dialysate during the dialysis interval 3, at screening and baseline visits, were required to have the serelaxin infusion was started after an interval of at least Cmax The observed maximum serum concentration following drug administration (mass/volume) CLfollowing: systolicThe systemic blood pressure(or total-body) (SBP) clearance ranging from from serum 110 following12 IV administration hours following (volume/time) the previous hemodialysis with PK

CLHDto 170� mm Hg,Hemodialysis diastolic clearance blood (volume/time)pressure (DBP) ranging sample collection and safety/tolerability assessments DEfrom 60 to 105Dialysis mm Hg, efficiency and pulse rate (PR) between 45 and being performed over the following 48 hours (until f100 bpm. TheFraction corresponding of drug eliminated inclusion during criteria dialysis for healthy day 3) and prior to the subsequent hemodialysis. All MRTsubjects were eGFRMean residence90 mL/(min time (time) 1.73 m2); matching in participants were required to remain at the study site QBINterms of race,Blood age ( flow�10 to years), the dialyzer sex,� (volume/time) and BMI ( 15%) to for approximately 3 days to facilitate baseline, PK, t Dialysis interval (time) an individual patient� with renal impairment in groups� 1, 2, and safety/tolerability assessments, with an additional Tmax Time to reach the maximum concentration after drug administration (time) or 3; SBP ranging from 100 to 150 mm Hg; DBP ranging ambulatory end-of-study visit on day 15. T1/2 The terminal elimination half-life (time) VDfrom 60 to 95 mmVolume Hg; of the and pooled PR between dialysate during 50 and the dialysis100 bpm. interval (volume)

Vz The keyThe exclusion volume of distribution criteria during were the terminal as follows: elimination phasePharmacokinetic following IV administration Assessments (volume) and Analyses V(1)ss clinically signiThe volumeficant ofelectrocardiogram distribution at steady abnormalities; state following IV administrationSerum (volume)samples for PK analysis were collected during the (2) history of hypersensitivity to the study drug or to treatment and follow-up periods (time points: pretreat- �As per the protocol, CLHD was calculated as (CD VD)/AUCt_in (Method 1). However, to avoid a potential underestimation because of adsorption, and drugs of a similar class; (3) use of other� investigational ment, 15 minutes, and 1, 2, 3, 4, 4.25, 5, 6, 7, 8, 9, 10, 11, 12, subsequent under-recovery of serelaxin in the dialysate, an alternative calculation was performed where CLHD QBIN R f, where R is the blood-to-serum drugs at the time of study enrollment; (4) presence of any 16, 24, 28, 36, and 48¼ hours� after� the start of infusion) and drug concentration ratio, which was assumedfi to be 55% (equivalent to the hematocrit as serelaxin does not bindfi to blood cells). IV, intravenous; PK,noncontrolled pharmacokinetics. and clinically signi cant disease, surgical, on day 15 (end of study). Speci cally for group 2 patients, or medical condition that could have affected the study who had hemodialysis performed from 6 to 10 hours post– outcome or that would have placed the patient at an undue serelaxin administration (4-hour dialysis period), serum serumrisk, as concentration judged by the following investigator; drug administration and (5) baseline (Cmax and). theirsamples intensity, for PK analysesand their were relationship collected to from the both study the drug line Inother addition, laboratory hemodialysis parameters clearance at screening (CLHD) outside was accept- calcu- wereentering evaluated (LINE for IN) all and participants. that exiting (LINE OUT) the latedable aslimits (CD asVD)/AUC judged byt_in the Method investigator. 1, where In CD addition, is the dialyzer. In addition, hemodialysis fluid samples were concentrationpatients with� of severe the drug renal in impairment/ESRD pooled dialysate during were Immunogenicitycollected from these Assessments patients 6 and hours Analyses prior to the start of theexcluded dialysis from interval, the study VD is if the they volume had ofhemoglobin pooled dialysate levels Serumdialysis and samples as fractions for immunogenicity during the 4-hour analysis dialysis period were during<9.0 g/dL the dialysis at screening interval and and received AUCt_in treatment is the area with under any collectedfor assesment before of theserelaxin start of concentrations. treatment and The on dialysate day 15 thecytostatic serum drug concentration-time or nitrate. Healthy curve subjects during were the excluded dialysis (endwas collected of study). in Antiserelaxin 20-L bottles,with antibodies 20-mL were aliquots evaluated (or 0.1% in intervalif they had on the used basis any of prescription the samples collected drugs within at the 4 entry weeks of serumof the inrespective a 4-tiered fraction) approach being with taken validated from assays.each bottle, The theprior dialyzer. to the To initial avoid a dosing potential and/or underestimation over-the-counter due to detailspooled, of and these stored assays at 3°C have to 5°C been for described the duration previously. of dialysis.7,8 adsorptionmedications and within subsequent 2 weeks underrecovery prior to the of initial serelaxin dosing, in SamplesAt the end were of initially the procedure, screened for 3-mL potential samples immunoge- of the theor had dialysate, tested positive an alternative for hepatitis calculation B or C.(Method 2) was nicitythoroughly in a screening mixed pooled assay. dialysate Any positive aliquots screening were results frozen performedPatients where in groups CLHD 1 to 3QBIN were enrolledR f, where in parallel QBIN into is requiredat 70°C con andfirmation later shipped using on an dry immunodepletion ice for analysis. assay. the bloodstudy.fl Healthyowing into subjects the¼ dialyzer; were� matched� R is the pairwise blood-to- and If� aThe sample serum was serelaxin immune concentrations depleted, it were was determined considered serumenrolled drug after concentration the corresponding ratio, which patient was assumed with torenal be positiveby a validated, and the samplecommercially would thenavailable move enzyme-linked to a third-tier 0.55impairment/ESRD as serelaxin does had not completed bind to serelaxin blood cells; treatment and f titrationimmunosorbent assay. In assay addition, (ELISA) any kit con (R&Dfirmed Systems, positive isand the PK fraction blood sample of drug collection. eliminated during hemodialysis, samplesMinneapolis, required Minnesota). testing for Brie neutralizationfly, the ELISA of serelaxin method calculatedStudy participants as (AUCt_in receivedAUC a singlet_out)/AUC open-label,t_in, 4-hourwhere biologicalused a monoclonal activity using antibody a validated specific cell-based for relaxin-2 bioassay. as the AUCIV infusiont_out is of the 10 areamg/kg under� serelaxin the serum on concentration-timeday 1. This dosing Ifcapture the sample reagent was and not an con enzyme-linkedfirmed as being polyclonal positive in anti- the curveregimen during (instead the dialysis of 30 mg/kg/day interval on for the 48 basis hours of that the immunodepletionbody specific for relaxin-2 assay, the as titration the detection and neutralization reagent. The sampleswas implemented collected at in the the exit serelaxin of the dialyzer.clinical development analyseslower limit were of not quanti performed.fication was 15.6 pg/mL. The same studies, including the ongoing phase 3 RELAX-AHF2 kit was used to determine the serelaxin concentration in Safetystudy) Assessments was chosen due and to Analyses the feasibility challenges with Statisticalthe dialysate Analyses for group 2, following validation, to ensure Safetyregard assessmentsto the timing included of PK regular assessments monitoring and the of Ano sample matrix size interference of 6 patients to in the each assay. of the The renal lower impairment limit of hematology,intermittent hemodialysis blood chemistry, (every coagulation, other day) and and urinalysis, for safety groupsquantifi matchedcation in with the dialysate 18 healthy was subjects 31.3 pg/mL. was calculated performedconsiderations. at a local A short laboratory; infusion andwas regular selected assessments over an IV to haveNoncompartmental 89% power to PKdetect analysis an observed was performed ratio of 2-fold using ofbolus body to minimize weight, vital the potential signs, physical risks associated condition, with and a changeWinNonlin at 10%Phoenix level (Version of signi 6.2).ficance, The evaluated assuming PK an electrocardiography.high initial serum concentration Adverse events following (AEs) an were IV sought bolus. interparticipantparameters are coef describedficient of in variation Table 1. of The 40% key (based PK byFor nondirective patients with questioning ESRD undergoing of participants PK assessments during the on onparameters a prior study, to compare where a serelaxin40% coeffi exposurecient of variation between was the studythe day and of were hemodialysis, also recorded the dialysis if volunteered procedure by was the obtainedpatient groups for the and AUC). matched8 healthy subjects were area participantsstarted 2 hours between ( 15 minutes) visits or after through the end the of above-listedinfusion and underParticipant the serum demographics concentration-time (age, sex, curve ethnicity, (AUC) race,from safetylasted assessments. for approximately� The frequency 4 hours. of The serious dialysis AEs machines (SAEs), weight,time 0 to and infi height)nity (AUC and) baseline and the characteristics observed maximum were 1 478Dahlke et al. The Journal of Clinical Pharmacology / Vol 56 No 4 20165

Table 2. Demographics of the Study Population

All Patients Total Study Population Group 1 Group 2 Group 3 (Groups 1, 2, and 3; Group 4 (Groups 1, 2, 3, and 4; Parameters (n 6) (n 6) (n 6) n 18) (n 18) n 36) ¼ ¼ ¼ ¼ ¼ ¼ Age (mean SD), years 58.7 10.1 46.5 12.0 52.3 15.8 52.5 13.1 52.0 12.0 52.3 12.4 � � � � � � � Males, % 83.3 66.7 83.3 77.8 77.8 77.8 White, % 100 100 100 100 100 100 BMI (mean SD), kg/m2 27.9 4.0 25.5 2.0 26.2 6.1 26.6 4.2 26.0 2.8 26.3 3.5 � � � � � � �

Group 1, patients with severe renal impairment; group 2, patients with ESRD requiring hemodialysis and assigned to receive serelaxin infusion on the day of dialysis; group 3, patients with ESRD requiring hemodialysis and assigned to receive serelaxin infusion during the dialysis-free interval; and group 4, matched healthy subjects. BMI, body mass index; ESRD, end-stage renal disease; SD, standard deviation.

summarized (Table 2). Data from all the participants were completion of infusion, serum serelaxin concentrations used for PK and safety analyses. The primary statistical declined rapidly, with a mean terminal elimination half- analyses were performed on the following PK parameters life ranging from 6.5 to 8.8 hours (Figure 2). Patients of serelaxin: Cmax and AUC . Parameters were compared with severe renal impairment or ESRD demonstrated a between each renal impaired1 group and the respective moderate decrease (37%–52%) in systemic serelaxin matched healthy subjects. Log-transformed PK param- clearance and a slight increase (10%-22%) in the apparent eters were analyzed using a linear mixed-effects model, volume of distribution at steady state compared with the with the healthy subject group as a fixed effect and the matched healthy controls (Table 3). matching pair as a random effect. Least-squares means for Compared with the healthy subjects, serelaxin reached each group as well as the estimated difference between a higher Cmax in patients with renal impairment. The patients with renal impairment and respective matched healthy subjects with corresponding 90% confidence intervals (CI) on the log-scale were calculated. These estimates were back-transformed to obtain the ratio of geometric means (GMR) and the associated 90% CI for the comparison of the renal impaired group vs the matched healthy subjects. In addition, PK parameters (Cmax and AUC ) obtained on the day of hemodialysis and during the1 dialysis-free interval were compared between the 2 groups of patients with ESRD to evaluate the impact of hemodialysis on the PK of serelaxin, with the healthy subject group as a fixed effect. Safety parameters were analyzed descriptively.

Results Study Population and Demographics A total of 36 participants, 18 patients with renal impairment/ESRD (6 each in groups 1–3) and 18 matched healthy subjects (group 4), were enrolled into the study. All 36 participants completed the study, and their data were included in the PK, immunogenicity, and safety/ tolerability analyses. The demographics were generally well balanced across the patient and healthy subjects groups (Table 2). Figure 2. Arithmetic mean (SD) serum serelaxin concentration–time profiles by group. (A) Linear and (B) semilogarithmic views of serum Pharmacokinetics of Serelaxin in Patients With Severe serelaxin concentration-time profiles. Data are shown as arithmetic Renal Impairment and ESRD mean with SD in linear and semilogarithmic views for patients with severe renal impairment (G1), ESRD requiring hemodialysis (with PK Following the start of IV infusion, serum serelaxin assessment on the day of dialysis [G2] and during the dialysis-free period concentrations increased rapidly and reached peak [G3]) compared with healthy controls (G4). ESRD, end-stage renal concentrations at the end of the 4-hour infusion. On disease; G, group; PK, pharmacokinetics; SD, standard deviation. 6Dahlke et al. The Journal of Clinical Pharmacology / Vol XX No XX 20154793

Tablecreatinine 3. Statistical clearance Summary levels of Serum calculated Serelaxin according PK Parameters to per the Groupused were Fresenius 4008s (Dialysator FX80, surface area simplified Modification of Diet in Renal Disease formula 1.8 m2), Fresenius 5008s (Dialysator FX80, surface area Parameters Group 1 (n 6) Group 2 (n 6) Group 3 (n 6) Group 4 (n 18) (15 mL/[min 1.73 m2] estimated glomerular¼ filtration 1.8¼ m2), Fresenius 5008s¼ (Dialysator FX1000, surface¼ AUCrate [eGFR](ng h/mL)<� 30 mL/[min� 1.73144 m (13.0)2]). Patients in groups 2131 (12.5)area 2.2 m2), and Gambro173 (10.5) AK 200 ultra (Dialysator82.2 (13.3) 1 � � Candmax (ng/mL) 3 were included if they21.7 (13.4) had ESRD requiring20.5 (9.4)Polyflux P210H). For21.2 (18.1)ESRD patients with PK15.4 assess-(13.5) Themodialysis.1/2 (hours) The vital signs for7.8 (10.1) patients in groups 1 to8.1 (15.8)ments performed during8.8 (11.3) the dialysis-free6.4 interval, (22.4) C (mL/[h kg]) 69.7 (13.0) 76.3 (12.5) 57.7 (10.5) 122 (13.3) 3,L at screening� and baseline visits, were required to have the serelaxin infusion was started after an interval of at least Vss (mL/kg) 391 (13.3) 432 (21.9) 424 (22.2) 355 (20.7) following: systolic blood pressure (SBP) ranging from 110 12 hours following the previous hemodialysis with PK Vz (mL/kg) 787 (14.6) 886 (14.7) 730 (18.6) 1120 (21.1) MRTto 170 (hours) mm Hg, diastolic blood5.6 pressure (13.4) (DBP) ranging5.7 (24.8)sample collection and7.4 (15.9) safety/tolerability assessments2.9 (19.3) from 60 to 105 mm Hg, and pulse rate (PR) between 45 and being performed over the following 48 hours (until All100 values bpm. are The presented corresponding as geometric inclusionmeans (coeffi criteriacient of variation, for healthy %). day 3) and prior to the subsequent hemodialysis. All Groupsubjects 1, patients were with eGFR severe renal90 mL/(min impairment;1.73 group m 2,2 patients); matching with ESRD in requiringparticipants hemodialysis werewith PK required assessment onto the remain day of dialysis; at the group study 3, patients site withterms ESRD of requiring race, age hemodialysis ( �10 years), with PK sex, assessment� and BMI during ( the15%) dialysis-free to interval;for approximately and group 4, matched 3 healthy days subjects. to facilitate baseline, PK, fi AUC , area under the serum� concentration-time curve from 0� to in nity; Cmax, observed maximum serum concentration following drug administration; CL, an individual1 patient with renal impairment in groups 1, 2, and safety/tolerability assessments, with an additional systemic clearance from serum; ESRD, end-stage renal disease; MRT, mean residence time; PK, pharmacokinetics; T , terminal elimination half-life; V , volume or 3; SBP ranging from 100 to 150 mm Hg; DBP ranging ambulatory end-of-study1/2 visit on day 15. ss of distribution at steady state; Vz, volume of distribution during the terminal elimination phase following intravenous administration. from 60 to 95 mm Hg; and PR between 50 and 100 bpm. The key exclusion criteria were as follows: Pharmacokinetic Assessments and Analyses GMR(1) clinically (in comparison significant with electrocardiogram healthy subjects) abnormalities; for Cmax were SerumTo determine samples for whether PK analysis the PK were of serelaxin collected was during affected the 1.39(2) history (90%CI of 1.26, hypersensitivity 1.53), 1.42 (90%CI to the study 1.29, drug 1.56), or and to bytreatment the timing and follow-up of hemodialysis periods (time relative points: to serelaxin pretreat- 1.30drugs (90%CI: of a similar 1.18, class; 1.43) (3) use in of groups other investigational1, 2, and 3, administration,ment, 15 minutes, the and arithmetic 1, 2, 3, 4, mean 4.25, 5,serum 6, 7, 8,concentration- 9, 10, 11, 12, respectivelydrugs at the time (Table of study 4). Furthermore, enrollment; compared(4) presence with of any the time16, 24, pro 28,files 36, from and 48 both hours ESRD after groups the start (groups of infusion) 2 and and 3) matchednoncontrolled healthy and subjects, clinically patients signifi withcant renaldisease, impairment surgical, wereon day compared. 15 (end of As study). shown Speci infi Figurecally for 3, groupthe mean 2 patients, serum hador medical a higher condition AUC thatwith could the followinghave affected GMRs: the study 1.61 concentration-vs-timewho had hemodialysis properformedfiles for from both 6groups to 10 hoursoverlapped post– (90%CIoutcome 1.46, or that 1.77), would1 1.70 have (90%CI placed the 1.54, patient 1.87), at andan undue 2.15 atserelaxin all early administration time points prior (4-hour to 6 hours dialysis and period), diverged serum only (90%CIrisk, as judged 1.95, 2.36) by the for investigator; groups 1, 2, and and (5) 3, baseline respectively and aftersamples the startfor PK of analyseshemodialysis were in collected group 2 from patients, both wherein the line (Tableother laboratory 4). parameters at screening outside accept- serumentering serelaxin (LINE IN) concentrations and that exiting declined (LINE much OUT) faster the ableSerelaxin limits as PK judged differences by the were investigator. much smaller In addition, than duringdialyzer. the In 4-hour addition, hemodialysis hemodialysis periodfluid compared samples were with thosepatients among with patients severe with renal renal impairment/ESRD impairment. Patients were thecollected equivalent from 4-hourthese patients period 6 in hours group prior 3 patients. to the start Upon of withexcluded ESRD from undergoing the study if PK they assessments had hemoglobin during levels the completiondialysis and of as fractionsdialysis, duringthe terminal the 4-hour elimination dialysis phases period hemodialysis-free<9.0 g/dL at screening period and had received the treatment slowest withsystemic any werefor assesment again in of parallel serelaxin with concentrations. each other between The dialysate the 2 serelaxincytostatic drugclearance, or nitrate. followed Healthy by subjects patients were with excluded severe groups.was collected Statistical in 20-L analysis bottles, con withfirmed 20-mL that aliquots although (or 0.1%Cmax renalif they impairment had used any and prescription then by drugspatients within with 4 ESRD weeks wasof the similar respective for groups fraction) 2 and being 3 (GMR taken 0.97, from 90%CI each bottle, 0.83, undergoingprior to the PK initial assessments dosing on and/or the day over-the-counter of hemodialysis 1.12),pooled, a and 4-hour stored dialysis at 3°C to on 5°C the for day the of duration infusion of dialysis.reduced (meanmedicationsstandard within deviation 2 weeks [SD]: prior 58.0 to the5.92 initial mL/[h dosing,kg] theAt overallthe end exposure of the by procedure, 24% versus 3-mL infusion samples during of the inor had group� tested 3, positive 76.8 9.71 for hepatitismL/[h kg] B or� in C. group 2,� and dialysis-freethoroughly mixed period pooled (GMR dialysate for AUC aliquots0.76; 90%CI were frozen 0.67, 70.1Patients8.38 inmL/[h groups�kg] 1 into group3 were 1;� enrolled Figure 2 in and parallel Table into 3). 0.85;at 70°C Table and 5). later shipped on dry ice1 for analysis. the study.� Healthy� subjects were matched pairwise and �The serum serelaxin concentrations were determined enrolled after the corresponding patient with renal by a validated, commercially available enzyme-linked Tableimpairment/ESRD 4. Statistical Analysis had of completed Serum Serelaxin serelaxin PK Parameters, treatment Comparisonimmunosorbent of Patients With Renal assay Impairment (ELISA) (Groups kit 1, (R&D2, and 3) vs Systems, Matched Healthyand PK Subjects blood (Group sample 4) collection. Minneapolis, Minnesota). Briefly, the ELISA method Study participants received a single open-label, 4-hour used a monoclonal antibody specific for relaxin-2 as the Adjusted Geometric Means� IV infusion of 10 mg/kg serelaxin on day 1. This dosing capture reagent and an enzyme-linkedObserved Ratio ofpolyclonal Geometric Means, anti- Parametersregimen (instead of 30Patientmg/kg/day Group for 48Patient hours that Matchedbody speciHealthyfic Subject for relaxin-2 as thePatient/Healthy detection reagent. (90%CI)� The was implemented in the serelaxin clinical development lower limit of quantification was 15.6 pg/mL. The same Cmax (ng/mL) Group 1 21.7 15.6 1.39 (1.26–1.53) studies, including the ongoingGroup 2 phase 3 RELAX-AHF220.5 kit was14.4 used to determine the serelaxin1.42 (1.29 concentration–1.56) in study) was chosen due toGroup the feasibility 3 challenges21.2 with the dialysate16.3 for group 2, following1.30 validation, (1.18–1.43) to ensure AUCregard(ng toh/mL) the timing ofGroup PK 1 assessments144 and the no matrix89.2 interference to the assay.1.61 The (1.46 lower–1.77) limit of 1 � intermittent hemodialysis (everyGroup 2 other day) and131 for safety quantifi77.1cation in the dialysate was 31.31.70 (1.54pg/mL.–1.87) considerations. A short infusionGroup 3 was selected173 over an IV Noncompartmental80.6 PK analysis was2.15 (1.95 performed–2.36) using bolus to minimize the potential risks associated with a WinNonlin Phoenix (Version 6.2). The evaluated PK �highAll values initial are back-transformed serum concentration from the log-scale. following Log-transformed an IV bolus. PK parameterparameters data were analyzed are using described a linear mixed-effects in Table model, 1. with The subje keyct group PK as a fixed effect and matched pair as a random effect. GroupFor patients 1, patients with with severe ESRD renal undergoing impairment; group PK 2, assessments patients with ESRD on requiringparameters hemodialysis to with compare PK assessment serelaxin on the day exposure of dialysis; group between 3, patients the withthe ESRD day requiring of hemodialysis, hemodialysis with the PK dialysisassessment procedureduring the dialysis-free was interval;patient and groups group 4, matched and matched healthy subjects. healthy subjects were area

AUCstarted, area 2 hours under( the15 serum minutes) concentration-time after theend curve of from infusion 0 to infinity; and CI, conunderfidence theinterval; serum Cmax,concentration-time observed maximum serum curve concentration (AUC) following from 1 � druglasted administration; for approximately ESRD, end-stage 4 hours. renal disease; The dialysis PK, pharmacokinetics. machines time 0 to infinity (AUC ) and the observed maximum 1 480Dahlke et al. The Journal of Clinical Pharmacology / Vol 56 No 4 20167

severe AEs were reported during the study period, and none of the participants discontinued the study due to an AE. Overall, AEs of mild intensity were reported in 4 participants: 2 in predose and 2 in postinfusion. The former AEs were increased lipase and increased blood creatine phosphokinase, reported in 2 healthy subjects, and the latter included a mild increase in blood creatine phosphokinase in 1 patient in group 1 and headache in 1 healthy subject. Headache was the only AE suspected by the investigator to be related to the study drug. All of these AEs were reported to have resolved by the end of the study period. Changes in hematology or clinical chemis- try, none of which were clinically significant, were reported in few of the healthy subjects. No trends or systematic changes in SBP, DBP, and PR were observed across the groups with a single 4-hour IV infusion of 10 mg/kg serelaxin. There were small changes in SBP, DBP, and PR, which were not clinically significant. Importantly, no apparent differences were observed in any of these changes between patients with renal impairment and the matched controls (Figure 5). Figure 3. Arithmetic mean (SD) serum serelaxin concentration-time profiles for the ESRD groups requiring hemodialysis. (A) Linear and Immunogenicity (B) semilogarithmic views of serum serelaxin concentration-time Antiserelaxin antibodies were not detected in any of the profiles. Data are shown as arithmetic mean with SD in linear and participants on either day 1 (predose) or on day 15 (post– semilogarithmic views for patients with ESRD requiring hemodialysis serelaxin treatment). with PK assessment on the day of dialysis (G2) and during the dialysis- free period (G3). ESRD, end-stage renal disease; G, group; PK, pharmacokinetics; SD, standard deviation. Discussion Further, analysis of blood entering and leaving the This study was designed to evaluate the PK, safety, and dialyzer revealed that serum serelaxin concentrations tolerability of a single 4-hour IV infusion of 10 mg/kg were lower in the line exiting than in the line entering the serelaxin in patients with severe renal impairment and dialyzer (Figure 4). The fraction of serelaxin eliminated those with ESRD requiring hemodialysis compared with during the 4-hour hemodialysis period was estimated to matched healthy subjects, and to inform whether potential be approximately 30% (geometric mean 0.297). Based on serelaxin dosage adjustments are needed in patients with the 2 methods of calculation described in the Methods severe renal impairment. section, the geometric mean for CLHD was either The dosing regimen in this study was chosen based on 552 mL/h (Method 1) or 3020 mL/h (Method 2; Table 6). practical and safety considerations as mentioned earlier. Although the dose rate (60 mg/kg/day for a 10 mg/kg dose Safety and Tolerability infused over 4 hours) was higher than the nominal Overall, a single 4-hour IV infusion of 10 mg/kg serelaxin serelaxin clinical dose for patients with AHF (30 mg/kg/day), was well tolerated in all participants. No deaths, SAEs, or both dose rates are well within the linear range

Table 5. Statistical Analysis of the Effect of Timing of Hemodialysis on Serum Serelaxin PK Parameters in Patients With ESRD Undergoing PK Assessment on the Day of Dialysis (n 6; Group 2) vs Those Undergoing PK Assessment During Dialysis-Free Interval (n 6; Group 3) ¼ ¼

Adjusted Geometric Means�

Patient PK on Day the PK During the Observed Ratio of Geometric Means, Parameters Group of Dialysis Dialysis-Free Interval Dialysis/Dialysis-Free (90%CI)�

Cmax (ng/mL) ESRD 20.5 21.2 0.97 (0.83–1.12) AUC (ng h/mL) ESRD 131 173 0.76 (0.67–0.85) 1 �

�All values are back-transformed from the log-scale. Log-transformed PK parameter data were analyzed using a linear mixed-effects model with subject group as fixed effect and matched pair as random effect.

AUC , area under the serum concentration–time curve from 0 to infinity; CI, confidence interval; Cmax, observed maximum serum concentration following 1 drug administration; ESRD, end-stage renal disease; PK, pharmacokinetics. 8Dahlke et al. The Journal of Clinical Pharmacology / Vol XX No XX 20154813 creatinine clearance levels calculated according to the (10used were100 m Freseniusg/kg/day) 4008s for serelaxin (Dialysator PK FX80, based surface on existing area simplified Modification of Diet in Renal Disease formula data.1.8� m72Thus,), Fresenius the essential 5008s PK(Dialysator characteristics FX80, generated surface area in (15 mL/[min 1.73 m2] estimated glomerular filtration this1.8 m study2), Fresenius can be used 5008s to inform (Dialysator whether FX1000, or not there surface is a rate [eGFR] <� 30 mL/[min� 1.73 m2]). Patients in groups 2 needarea to 2.2 adjust m2), the and clinical Gambro dose AK for 200 patients ultra with (Dialysator AHF. and 3 were included if� they had ESRD requiring PolyOfflux note, P210H). serelaxin, For ESRD being patients a recombinant with PK peptide assess- hemodialysis. The vital signs for patients in groups 1 to identicalments performed to the endogenous during human the dialysis-free relaxin-2, is expected interval, 3, at screening and baseline visits, were required to have the toserelaxin be cleared infusion via the was same started pathways after an as interval the endogenous of at least following: systolic blood pressure (SBP) ranging from 110 relaxin.12 hours1,2,9 followingThe main the elimination previous hemodialysis pathway for serelaxin with PK to 170 mm Hg, diastolic blood pressure (DBP) ranging issample most likely collection to be via and catabolism safety/tolerability by peptidases/proteases assessments from 60 to 105 mm Hg, and pulse rate (PR) between 45 and throughoutbeing performed the body, over including the following the kidneys, 48 hours liver, (until and 100 bpm. The corresponding inclusion criteria for healthy otherday 3) organs and prior and tissues. to the9,10 subsequentGiven its hemodialysis. small molecular All subjects were eGFR 90 mL/(min 1.73 m2); matching in massparticipants (5.9 kDa), were serelaxin required is to expected remain toat be thefi studyltered site via terms of race, age ( �10 years), sex,� and BMI ( 15%) to glomerularfor approximatelyfiltration 3 in days the kidneys to facilitate and then baseline, metabolized PK, an individual patient� with renal impairment in groups� 1, 2, viaand intraluminal safety/tolerability metabolism assessments, or intracellular with an lysosomal additional or 3; SBP ranging from 100 to 150 mm Hg; DBP ranging metabolismambulatory end-of-study in the tubular visit on region day 15. of the kidneys. from 60 to 95 mm Hg; and PR between 50 and 100 bpm. Therefore, it is not surprising to see a slower clearance The key exclusion criteria were as follows: andPharmacokinetic higher exposure Assessments of serelaxin and in Analyses patients with severe (1) clinically significant electrocardiogram abnormalities; renalSerum impairment samples for or PK ESRD analysis when were compared collected with during matched the (2) history of hypersensitivity to the study drug or to healthytreatment subjects. and follow-up However, periods the differences (time points: are moderate, pretreat- drugs of a similar class; (3) use of other investigational withment, the 15 GMRminutes, of and AUC 1, 2,ranging 3, 4, 4.25, from 5, 6, 1.61 7, 8, to9, 10, 2.15. 11, The 12, drugs at the time of study enrollment; (4) presence of any moderate16, 24, 28, difference 36, and 48 is hours1 partly after due the to start the of contribution infusion) and of noncontrolled and clinically significant disease, surgical, theon day catabolism 15 (end of of study). serelaxin Speci infi othercally for tissues group and 2 patients, organs Figure 4. Arithmetic mean (SD) serum serelaxin concentration-time proorfi medicalles for the condition ESRD group that requiring could hemodialysis have affected and assignedthe study to outsidewho had the hemodialysis kidneys as wellperformed as the distributionfrom 6 to 10 of hours serelaxin post– receiveoutcome serelaxin or that infusion would on have the placedday of dialysis the patient—LINE at IN an and undue LINE intoserelaxin the extravascular administration space (4-hour (Vss dialysis355–432 period), mL/kg). serum This OUT.risk, (A)as judgedLinear view by and the (B) investigator; semilogarithmic and views (5) ofserum baseline serelaxin and issamples also consistent for PK analyses with the were observed collected¼ moderate from both impact the line of concentration-timeother laboratory pro parametersfiles. Data are at shownscreening as arithmetic outside mean accept- with hemodialysisentering (LINE on IN) the PK and of that serelaxin. exiting During (LINE the OUT) 4-hour the SDable in linear limits and as semilogarithmic judged by views the for investigator. patients with ESRD In addition, requiring hemodialysis,dialyzer. In addition, 30% of hemodialysis serum serelaxinfluid was samples eliminated were hemodialysis and assigned to receive serelaxin infusion on the day of dialysispatients (G2) with—LINEsevere IN and LINE renal OUT. impairment/ESRD ESRD, end-stage renal disease; were bycollected hemodialysis, from these which patients constituted 6 hours approximately prior to the start 52% of G,excluded group; SD, from standard the deviation. study if they had hemoglobin levels ofdialysis the and overall as fractions systemic during clearance, the 4-hour indicating dialysis period that <9.0 g/dL at screening and received treatment with any for assesment of serelaxin concentrations. The dialysate cytostatic drug or nitrate. Healthy subjects were excluded was collected in 20-L bottles, with 20-mL aliquots (or 0.1% Tableif they 6. hadSummary used Statistics any prescription for Dialysis in drugs Group within 2 (n 6) 4 of weeks Serelaxin PKof Parameters the respective fraction) being taken from each bottle, ¼ prior to the initial dosing and/or over-the-counter pooled, and stored at 3°C to 5°C for the duration of dialysis. medications within 2 weeks prior to the initial dosing, At the endStatistics of the procedure, 3-mL samples of the Parametersor had tested positive for hepatitis B orMean C. (SD) thoroughlyGeometric mixed Mean (CV%) pooled dialysate aliquotsMedian were (Min, frozen Max) Patients in groups 1 to 3 were enrolled in parallel into at 70°C and later shipped on dry ice for analysis. CLHDthe study. (mL/h)� Healthy subjects were matched619 (308) pairwise and �The serum552 (57.7) serelaxin concentrations were517 (242, determined 996) CLHD_2 (mL/h)�� 3210 (1260) 3020 (39.2) 2680 (2010, 5060) CLenrolled (mL/h) after the corresponding patient5870 (1130) with renal by a validated,5780 (19.1) commercially available5710 enzyme-linked (4360, 7800) CLHD/CLimpairment/ESRD (%) had completed serelaxin11.2 (6.9) treatment immunosorbent9.56 (69.2) assay (ELISA) kit (R&D9.24 (4.13, Systems, 22.7) CLHD_2/CLand PK blood (%) sample collection. 57.5 (29.3) Minneapolis,52.2 (50.0) Minnesota). Briefly, the42.8 ELISA (32.3, 102)method F Study participants received a single0.312 open-label, (0.108) 4-hour used a monoclonal0.297 (34.9) antibody specific for0.271 relaxin-2 (0.203, 0.452) as the DEIV† infusion of 10 mg/kg serelaxin on0.034 day (0.0176) 1. This dosing capture0.0299 reagent (63.9) and an enzyme-linked0.0287 polyclonal (0.0115, 0.0554) anti- †† DE_2regimen (instead of 30 mg/kg/day0.171 for (0.0596) 48 hours that body speci0.163fic (34.9) for relaxin-2 as the detection0.149 reagent.(0.111, 0.248) The AUC (ng h/mL) 20.1 (2.93) 19.9 (15.6) 20.4 (15.2, 23.6) wast_in implemented� in the serelaxin clinical development lower limit of quantification was 15.6 pg/mL. The same AUC (ng h/mL) 13.8 (2.71) 13.6 (19.5) 13.2 (10.6, 17.9) t_out � Amountstudies, dialyzed including (ng) the ongoing phase12 200 3 RELAX-AHF2 (5880) kit was11 used 000 (56.4)to determine the serelaxin11 concentration 100 (4560, 22 000) in Bloodstudy) flow was rate chosen (mL/min) due to the feasibility308.3 challenges (20.41) with the dialysate307.8 (6.3)for group 2, following validation,300 (300, to 350) ensure Rateregard of dialysis to (mL/min)the timing of PK assessments445 (68.63) and the no matrix440.3 interference (16.2) to the assay. The475 lower (360, 500) limit of intermittent hemodialysis (every other day) and for safety quantification in the dialysate was 31.3 pg/mL. �considerations.CLHD = amount dialyzed/AUC A short infusiont in. was selected over an IV Noncompartmental PK analysis was performed using ��CLHD_2 = blood flow into the dialyzer 55% f. bolus to minimize the potential� risks� associated with a WinNonlin Phoenix (Version 6.2). The evaluated PK † fl highDE = CLHD/blood initial serumow intoconcentration the dialyzer. following an IV bolus. parameters are described in Table 1. The key PK †† fl ForDE_2 patients = CLHD_2/blood with ESRDow into undergoing the dialyzer. PK assessments on parameters to compare serelaxin exposure between the Group 2: patients with ESRD requiring hemodialysis and assigned to receive serelaxin infusion on the day of dialysis. AUCthet dayin/out, of AUC hemodialysis, during dialysis interval the (t) dialysis for serum samples procedure collected was at the entry/exitpatient of the groups dialyzer; and CL, systemic matched clearance; healthy CLHD, subjects hemodialysis were clearance area started 2 hours ( 15 minutes) after the end of infusionfi and under the serum concentration-time curve (AUC) from (calculated using two� methods as CLHD and CLHD_2); CV, coef cient of variation; DE, dialyzer extraction ratio (calculated using two methods as shown belowlasted as for DE and approximately DE_2); ESRD, end-stage 4 hours. renal The disease; dialysis f, fraction machines eliminated by dialysis;time 0 PK, to pharmacokinetics; infinity (AUC SD,) standard and the deviation. observed maximum 1 482Dahlke et al. The Journal of Clinical Pharmacology / Vol 56 No 4 20169

impact and relevance of the observed PK differences in this study, a population PK analysis was performed on the basis of the pooled data from subjects, including patients with AHF, with various degrees of renal function (normal, mild, moderate, and severe impairment and ESRD) who were treated with serelaxin across multiple studies. Data from this analysis showed that serelaxin clearance was reduced by 38% and 47% in patients with ESRD compared with patients with mild renal impairment and healthy subjects with normal renal function, respectively (data on file). Therefore, serelaxin PK differences between patients with severe renal impairment or ESRD and patients with mild to moderate renal impairment are smaller than the differences observed in this study when healthy subjects with normal renal function are used as comparators. In addition, clinical data suggest that serelaxin has a wide therapeutic window and a shallow exposure/dose- response relationship.4,11 Serelaxin doses of up to 960 mg/kg/day were administered to patients with AHF, and it was demonstrated that doses ranging from 10 to 100 mg/kg/day were generally well tolerated in patients with AHF.4,11 Moreover, serelaxin was well tolerated in this study by all subjects, including patients with ESRD, regardless of their increased exposure to serelaxin. The PK differences observed in this study are not likely to pose a safety risk to patients with severe renal impairment or ESRD and do not warrant a predefined dosage adjustment based on renal function. Possible limitations of this study are that it did not evaluate the effect of peritoneal dialysis on the PK of serelaxin, a factor that may be of particular relevance for Figure 5. Arithmetic mean (SD) values over time per group for patients with ESRD requiring dialysis. Second, the effect (A) sitting systolic blood pressure, (B) sitting diastolic blood pressure, and (C) pulse rate. Data are shown as arithmetic mean with SD of dialysis on the PK of serelaxin was evaluated based on for patients with severe renal impairment (G1), ESRD requiring the comparison between the 2 parallel groups of patients hemodialysis (with PK assessment on the day of dialysis [G2] and during (groups 2 and 3), and no intragroup comparisons were the dialysis-free period [G3]) compared with healthy controls (G4). performed, where PK data are collected before and during BAS, baseline; BP, blood pressure; ESRD, end-stage renal disease; dialysis from the same patients. Given the observed small G, group; SD, standard deviation. interpatient serelaxin PK variability, and considering the serious health state of the patient population, the parallel- group design is a sound approach for the purpose of the hemodialysis accelerates drug clearance from the body. study. Two different methods were used to calculate the In summary, this study demonstrated a moderate estimated CLHD. Method 1 used the total amount of decrease in clearance and an increase in exposure of serelaxin recovered from the dialysate in the calculation serelaxin in patients with severe renal impairment or and generated a lower value than the CLHD estimated ESRD compared with healthy subjects. It also showed using Method 2, which by definition is independent of the that serelaxin can be partially eliminated from circulation serelaxin concentration measurement in the dialysate. The by hemodialysis. Serelaxin was well tolerated by patients discrepancy between these results was most likely due to with severe renal impairment, by those with ESRD underestimation of the total amount of serelaxin in the requiring hemodialysis, and by healthy subjects. No dialysate, potentially through adsorptive loss of serelaxin antiserelaxin antibodies were detected in any participant. to the dialysis device and tubing. Therefore, the results The observed serelaxin PK differences in patients with provided by Method 2 are considered more reliable. Renal severe renal impairment compared with matched healthy impairment and dysfunction are often noted in patients subjects are unlikely to pose a safety risk and do not with HF.3,4,6 Therefore, in order to evaluate the clinical warrant a predefined dosage adjustment in such patients. 10Dahlke et al. The Journal of Clinical Pharmacology / Vol XX No XX 20154833

Acknowledgmentscreatinine clearance levels calculated according to the used3. Teerlink were Fresenius JR, Cotter G, 4008s Davison (Dialysator BA, et al. FX80,Serelaxin, surface recombinant area fi fi human2 relaxin-2, for treatment of acute heart failure (RELAX-AHF): Thesimpli authorsed Modi acknowledgecation of Ruchika Diet in Srinivasan, Renal Disease Roohi formula Chopra 1.8 m ), Fresenius 5008s (Dialysator FX80, surface area 2 fi a randomised,2 placebo-controlled trial. Lancet. 2013;381(9860): (Global(15 mL/[min Medical1.73 & Clinical m ] Services,estimated Novartis glomerular Healthcareltration Pvt. 1.8 m ), Fresenius 5008s (Dialysator FX1000, surface � � 2 29–39. 2 Ltd.,rate [eGFR] Hyderabad,<30 India), mL/[min and1.73 Paul m Coyle]). Patients (Global in Medical groups & 2 area4. Teerlink 2.2 m JR,), Metra and M, Gambro Felker GM, AK et al. 200 Relaxin ultra for the (Dialysator treatment of � fl Clinicaland 3 Services, were included Novartis if Ireland they Ltd.) had for ESRD providing requiring their Polypatientsux P210H). with acute heart For failure ESRD (Pre-RELAX-AHF): patients with PKa multicentre, assess- fi medicalhemodialysis. writing The support. vital signs for patients in groups 1 to mentsrandomised, performed placebo-controlled, during the parallel-group, dialysis-free dose- nding interval, phase IIb study. Lancet. 2009;373(9673):1429–1439. 3, atAt screening all stages and the baseline authors have visits, had were control required over tothe have content the serelaxin infusion was started after an interval of at least following: systolic blood pressure (SBP) ranging from 110 125. Smithhours GL, following Lichtman JH, the Bracken previous MB, hemodialysis et al. Renal impairment with PKand of this manuscript; have contributed to drafting the manuscript, outcomes in heart failure: systematic review and meta-analysis. reviewing,to 170 mm and Hg, revising diastolic it critically blood pressure for important (DBP) intellectual ranging sampleJ Am Coll collection Cardiol. 2006;47(10):1987 and safety/tolerability–1996. assessments content;from 60 to and 105 have mm providedHg, and pulsefinal rate approval (PR) between of the version 45 and being6. Heywood performed JT, Fonarow over GC, the Costanzo following MR, Mathur 48 hours VS, Wignes- (until submitted100 bpm. for The publication. corresponding inclusion criteria for healthy daywaran 3) and JR, Wynne prior J. to High the prevalence subsequent of renal hemodialysis. dysfunction and All its subjects were eGFR 90 mL/(min 1.73 m2); matching in participantsimpact on outcomewere required in 118,465 to patients remain hospitalized at the study with acute site Fundingterms of race, and age Declaration ( �10 years), of sex, Con� andflicting BMI Interests ( 15%) to fordecompensated approximately heart 3 failure: days a report to facilitate from the ADHERE baseline, database. PK, � � J Card Fail. 2007;13(6):422–430. Thisan individual study was patient sponsored with renal by Novartis impairment Pharma in groups AG, Basel, 1, 2, and7. Dahlke safety/tolerability M, Ng D, Yamaguchi assessments, M, et al. Safety with and an tolerability additional of Switzerland.or 3; SBP ranging M.D., J.C., from and 100 Y.P. to are 150 employees mm Hg; and DBP stockholders ranging ambulatoryserelaxin, a end-of-study recombinant human visit relaxin-2, on day in15. development for the offrom Novartis. 60 to 95 A.H. mm received Hg; and remuneration PR between for 50 participating and 100 bpm. as treatment of acute heart failure, in healthy Japanese volunteers and a comparison of pharmacokinetics and pharmacodynamics in healthy an investigatorThe key for exclusion the study. criteriaS.M. is a full-timewere as employee follows: of Pharmacokinetic Assessments and Analyses (1) clinically significant electrocardiogram abnormalities; SerumJapanese samples and Caucasian for PK analysis populations. wereJ collectedClin Pharmacol during. 2015; the Novartis Healthcare Pvt Ltd. C.T. was a full-time employee of 55(4):415–422. Novartis(2) history at the of hypersensitivitytime of the study to and the development study drug of or this to treatment8. Kobalava and Z, Villevalde follow-up S, Kotovskaya periods Y, (time et al. points: Pharmacokinetics pretreat- of manuscriptdrugs of a and similar has left class; the company(3) use of at other the time investigational of submitting ment,serelaxin 15 minutes, in patients and with 1, hepatic 2, 3, impa4, 4.25,irment: 5, a 6, single-dose, 7, 8, 9, 10, open-label, 11, 12, thisdrugs manuscript. at the time of study enrollment; (4) presence of any 16,parallel-group 24, 28, 36, andstudy. 48Br hours J Clin Pharmacol after the. start 2014;79(6): of infusion) 937–945. and noncontrolled and clinically significant disease, surgical, on9. day Bennett 15 (end RG, Heimann of study). DG, Speci Hamelfically FG. for Degradation group 2 ofpatients, relaxin or medical condition that could have affected the study whofamily had hemodialysis peptides by insulin-degrading performed from enzyme. 6 toAnn 10 hoursNY Acad post Sci–. References 2009;1160:38–41. outcome or that would have placed the patient at an undue serelaxin administration (4-hour dialysis period), serum 1. Teichman SL, Unemori E, Teerlink JR, Cotter G, Metra M. Relaxin: 10. Cossum PA, Dwyer KA, Roth M, et al. The disposition of a human risk,review as judged of biology by and the potential investigator; role in treating and (5) heart baseline failure. Curr and samplesrelaxin for (hRlx-2) PK analyses in pregnant were and collected nonpregnant from rats. bothPharm the lineRes. otherHeart laboratory Fail Rep. 2010;7(2):75parameters–82. at screening outside accept- entering1992;9(3):419 (LINE–424. IN) and that exiting (LINE OUT) the able2. Du limits X-J, Bathgate as judged RAD, by Samuel the investigator. CS, Dart AM, In Summers addition, RJ. 11.dialyzer. Dschietzig In T, addition, Teichman S, hemodialysis Unemori E, et al.fl Intravenousuid samples recombinant were patientsCardiovascular with severeeffects of renal relaxin: impairment/ESRD from basic science to clinical were collectedhuman relaxin from in these compensated patients heart 6 hoursfailure: aprior safety, to tolerability, the start and of pharmacodynamic trial. J Card Fail. 2009;15(3): 182–190. excludedtherapy. fromNat Rev the Cardiol study. 2010;7(1):48 if they had–58. hemoglobin levels dialysis and as fractions during the 4-hour dialysis period <9.0 g/dL at screening and received treatment with any for assesment of serelaxin concentrations. The dialysate cytostatic drug or nitrate. Healthy subjects were excluded was collected in 20-L bottles, with 20-mL aliquots (or 0.1% if they had used any prescription drugs within 4 weeks of the respective fraction) being taken from each bottle, prior to the initial dosing and/or over-the-counter pooled, and stored at 3°C to 5°C for the duration of dialysis. medications within 2 weeks prior to the initial dosing, At the end of the procedure, 3-mL samples of the or had tested positive for hepatitis B or C. thoroughly mixed pooled dialysate aliquots were frozen Patients in groups 1 to 3 were enrolled in parallel into at 70°C and later shipped on dry ice for analysis. the study. Healthy subjects were matched pairwise and �The serum serelaxin concentrations were determined enrolled after the corresponding patient with renal by a validated, commercially available enzyme-linked impairment/ESRD had completed serelaxin treatment immunosorbent assay (ELISA) kit (R&D Systems, and PK blood sample collection. Minneapolis, Minnesota). Briefly, the ELISA method Study participants received a single open-label, 4-hour used a monoclonal antibody specific for relaxin-2 as the IV infusion of 10 mg/kg serelaxin on day 1. This dosing capture reagent and an enzyme-linked polyclonal anti- regimen (instead of 30 mg/kg/day for 48 hours that body specific for relaxin-2 as the detection reagent. The was implemented in the serelaxin clinical development lower limit of quantification was 15.6 pg/mL. The same studies, including the ongoing phase 3 RELAX-AHF2 kit was used to determine the serelaxin concentration in study) was chosen due to the feasibility challenges with the dialysate for group 2, following validation, to ensure regard to the timing of PK assessments and the no matrix interference to the assay. The lower limit of intermittent hemodialysis (every other day) and for safety quantification in the dialysate was 31.3 pg/mL. considerations. A short infusion was selected over an IV Noncompartmental PK analysis was performed using bolus to minimize the potential risks associated with a WinNonlin Phoenix (Version 6.2). The evaluated PK high initial serum concentration following an IV bolus. parameters are described in Table 1. The key PK For patients with ESRD undergoing PK assessments on parameters to compare serelaxin exposure between the the day of hemodialysis, the dialysis procedure was patient groups and matched healthy subjects were area started 2 hours ( 15 minutes) after the end of infusion and under the serum concentration-time curve (AUC) from lasted for approximately� 4 hours. The dialysis machines time 0 to infinity (AUC ) and the observed maximum 1