A Randomized, 29-Day, Dose-Ranging, Efficacy and Safety Study of Daprodustat, Administered Three Times Weekly in Patients with Anemia on Hemodialysis Christine K

Bailey et al. BMC Nephrology (2019) 20:372 https://doi.org/10.1186/s12882-019-1547-z

RESEARCH ARTICLE Open Access A randomized, 29-day, dose-ranging, efficacy and safety study of daprodustat, administered three times weekly in patients with anemia on hemodialysis Christine K. Bailey1*, Stephen Caltabiano2, Alexander R. Cobitz1, Chun Huang3, Kelly M. Mahar4 and Vickas V. Patel5

Abstract Background: Daprodustat is a hypoxia-inducible factor-prolyl hydroxylase inhibitor currently being investigated as a treatment for anemia of chronic kidney disease (CKD) in both dialysis and nondialysis patients. In clinical studies to date, daprodustat has been administered orally as a once-daily regimen. This randomized, double-blind, placebo-controlled study characterized the initial dose-hemoglobin response as well as the efficacy and safety of three times weekly (TIW) daprodustat in hemodialysis patients switched from stable recombinant human erythropoietin (rhEPO), in accordance with a TIW hemodialysis schedule. Methods: 103 patients on hemodialysis with baseline hemoglobin of 9.0 to 11.5 g/dL and previously receiving a stable dose of rhEPO or its analogs were randomized 1:1:1:1:1 to receive daprodustat 10, 15, 25, or 30 mg or placebo TIW over 29 days. Results: Mean baseline hemoglobin was 10.6 g/dL for the placebo group and each daprodustat cohort. Daprodustat produced dose-dependent changes in mean hemoglobin from baseline to day 29. Using a Bayesian approach, the estimated dose conversion ratio between once-daily and TIW daprodustat was ~ 2.0 across the evaluated dose range using an Emax model. Daprodustat was generally well tolerated, with an adverse event (AE) profile consistent with the hemodialysis population. Conclusions: These data help inform the appropriate dose conversion ratio to be applied to daily doses to obtain equivalent daprodustat TIW doses and suggest TIW treatment with daprodustat can treat anemia of CKD safely, supporting future long-term studies for this indication using a TIW dosing regimen. Trial registration: ClinicalTrials.gov Identifier: NCT02689206; date registered: 02/11/2016. Keywords: Anemia, Hemodialysis, Three-times weekly (TIW), Dose response, Bayesian, Efficacy, Clinical trials, Safety

Background While effective therapies are available for anemia of CKD, The causes of anemia in patients with chronic kidney dis- including supplemental iron, recombinant human EPO ease (CKD) are multi-factorial, including relative or abso- (rhEPO) and its analogs, and blood transfusions, each has lute deficiency of erythropoietin (EPO), reduced iron significant limitations [3–5]. Specifically, adverse cardio- availability related to chronic inflammation, and gastro- vascular events have been reported with rhEPO and its an- intestinal blood loss [1, 2]. Anemia is further exacerbated alogs, which led the US Food and Drug Administration to by shortened erythrocyte survival, which is associated with limit use of these treatments in patients on hemodialysis the uremic milieu and the hemodialysis procedure [1]. with hemoglobin levels < 10 g/dL, and the dose should be reduced or the drug discontinued when hemoglobin ex- * Correspondence: [email protected] ceeds 11 g/dL [3]. 1R&D, Clinical Sciences, GlaxoSmithKline, 1250 S. Collegeville Road, Mail Code Hypoxia-inducible factor (HIF)-prolyl hydroxylase in- UP 4200, Collegeville, PA 19426, USA hibitors (PHIs) are an emerging new class of therapy for Full list of author information is available at the end of the article

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bailey et al. BMC Nephrology (2019) 20:372 Page 2 of 12

the treatment of anemia of CKD. These molecules stimu- The dose-response relationship for once-daily dos- late erythropoiesis through inhibition of HIF-prolyl ing of daprodustat in the hemodialysis population was hydroxylase domain enzymes (PHD1, PHD2, PHD3) established in a 24-week phase 2 study (ClinicalTrials. resulting in the accumulation of HIFα transcription fac- gov identifier NCT01977482) [9]. In that study, the tors and increased expression of HIF-responsive genes hemoglobin response (change from baseline) was de- under normoxic conditions [6]. HIF-responsive genes termined at 4 weeks over the dose range of 4 mg to 12 include EPO and other proteins involved in increasing mg; therefore, in order to make a direct comparison oxygen availability and utilization as well as proteins between the once-daily and TIW regimens, a 29-day involved in iron utilization, angiogenesis, apoptosis, me- treatment period was utilized. tabolism, and vascular tone [7]. Based on their mechanism The study was initiated on February 17, 2016 and of action, HIF-PHIs are postulated to be associated with completed on January 25, 2017. Twenty-nine centers in fewer major adverse cardiovascular events by raising 5 countries randomized at least one participant: 8 cen- hemoglobin without the supraphysiologic EPO concentra- ters in the Russian Federation, 7 each in Spain and the tions associated with rhEPO therapy [6]. United States, 4 in Canada, and 3 in Germany. Daprodustat is a HIF-PHI currently being investi- Study participants were stratified by low or high prior gated as a treatment for anemia of CKD in both dialysis dose of rhEPO (or its analogs) based on the average and non-dialysis patients [6, 8]. In clinical studies to weekly dose during the 12 weeks prior to randomization date, daprodustat has been administered orally as a (day 1). Low dose was defined as < 100 IU/kg/week epoe- once-daily regimen [8, 9]. However, the preferred prac- tin or < 0.5 μg/kg/week darbepoetin. High dose was de- tice in countries that utilize a three times weekly (TIW) fined as ≥100 IU/kg/week epoetin or ≥ 0.5 μg/kg/week hemodialysis schedule is to administer anemia therapy darbepoetin. Participants were randomized in equal pro- at the time of the hemodialysis session [10]. portions to a daprodustat dose or placebo within each The rationale of this study was to assess the feasibility of stratum without using prior rhEPO dose to determine administering daprodustat in conjunction with a TIW the daprodustat dose. hemodialysis schedule, and determine the equivalent TIW Participants discontinued their current rhEPO therapy doses of daprodustat required to maintain hemoglobin prior to day 1 and were randomized by a computer- levels in patients previously stable and responding to ther- generated randomization schedule using the Prism apy with rhEPO or its analogs who are switched to dapro- randomization system in a 1:1:1:1:1 ratio to receive oral dustat. The primary objective was to characterize the daprodustat 10, 15, 25, or 30 mg or placebo TIW on a initial hemoglobin dose-response relationship for TIW dialysis day. Evidence with epoetin alfa (Epogen), has dosing of daprodustat at day 29 and to describe the rela- shown that increases in dosing interval can be achieved tionship between once-daily and TIW dosing of daprodu- by a proportionate increase in the dose administered at stat in hemodialysis-dependent patients with anemia of each interval [11]. As the daprodustat dose response CKD who are switched from a stable dose of rhEPO or its with hemoglobin was generally linear between 4 and 24 analogs. Additional objectives included estimation of the mg once daily, it was anticipated that a similar time- dose conversion ratio between once-daily and TIW dapro- proportional efficacy relationship would exist for daprodustat; characterization of the pharmacodynamic (PD) dustat and the management of hemoglobin levels would effect of daprodustat TIW dose regimens on EPO, vascu- remain as the dosing was extended from once every day lar endothelial growth factor (VEGF), markers of iron to three times weekly. Based on the hypothesis that the metabolism, and indices of hematopoiesis; and assessment total weekly TIW dose that achieves the same total of the pharmacokinetics (PK), safety, and tolerability of weekly once-daily dose would have the same Hgb effect, daprodustat TIW. the TIW dose would be equivalent to ~ 2.3 times the once-daily dose (7 doses/ week × 1 week/ 3 doses = 2.3 multiplier), doses of 10, 15, 25, and 30 mg TIW were in- Methods vestigated for 29 days to most closely match the 4, 6, 10, Study design and 12 mg once-daily doses that were evaluated in the This was a randomized, double-blinded, dose-ranging, study by Meadowcroft et al. [9]. Studying multiple doses placebo-controlled, parallel group study. The study con- allowed a robust analysis of a range of TIW doses to de- sisted of a 28-day screening period, a 29-day treatment termine the “equivalent” ratio to bridge to once-daily period (with study visits on days 1, 15, and 29), and a dose levels. The dose-hemoglobin response relationship follow-up visit approximately 14 days after completing was generally linear in the once-daily dose range of treatment. For patients on dialysis TIW, the day 1, 15, interest (4 to 24 mg) but it was not known if this was and 29 study visits could not occur on the first dialysis maintained with the higher doses needed for TIW session following the longest intradialytic interval. dosing. Bailey et al. BMC Nephrology (2019) 20:372 Page 3 of 12

Each participant was assigned a randomization number cancer, or a known complex kidney cyst (ie, Bosniak by the interactive voice and web response system, which Category II F, III, or IV) > 3 cm. was managed by Pharmaceutical Product Development, LLC, Wilmington, NC, USA. Once a randomization num- Withdrawal criteria ber was assigned, it could not be reassigned. Study partici- Study medication was permanently discontinued if participants, investigators, and site and sponsor staff were all pants met the hemoglobin stopping criteria, based on blinded to the treatment assignment. To maintain the HemoCue measured hemoglobin values as follows: a con- blind, each participant took one tablet of study treatment firmed hemoglobin < 7.5 g/dL, a confirmed hemoglobin in- from each of three bottles of study treatment, on each dos- crease ≥1.0 g/dL over the previous 2 weeks, or a confirmed ing day. The combination of tablets provided the appropri- hemoglobin decrease ≥2.0 g/dL over the previous 2 weeks ate dose of study treatment, and included a combination of based on point of care hemoglobin measurements. For con- tablet strengths, active and placebo, to correspond to the firmed hemoglobin ≥13.0 g/dL, a decision regarding with- appropriate dose. An internal Safety Review Team (SRT), drawal was made in consultation with the GlaxoSmithKline which included GlaxoSmithKline personnel involved in the Medical Monitor. Central lab hemoglobin values were also conduct of the study, monitored blinded safety data in- analyzed retrospectively to identify how many participants stream during the study. met each of the hemoglobin stopping criteria. Participants were also permanently discontinued from Study population study treatment if they received a blood transfusion or ndividuals who were at least 18 years old and had stable kidney transplant, became or intended to become preg- baseline hemoglobin values between 9.0 g/dL and 11.5 g/dL nant, had active GI bleeding, were diagnosed with cancer based on average values obtained on day − 28, day − 14, (with the exception of localized squamous cell and basal and day 1 were eligible for the study. Study participants cell carcinoma), or missed 2 consecutive dialysis sessions. were also required to be on a stable hemodialysis regimen Liver chemistry was also monitored during the study. of 3 to 5 times weekly for at least 90 days, with a single- Participants who discontinued study treatment com- pool Kt/Vurea of ≥1.2. Additional eligibility criteria included pleted an early withdrawal assessment and follow-up that participants be on a stable dose of rhEPO or its ana- visit 14 ± 3 days after the last dose of study treatment. logs, with the total weekly dose varying by no more than 50%, during the 4 weeks prior to day − 28. A protocol Study analyses amendment on May 11, 2016 updated the eligibility criteria No formal sample size or power calculations were per- to include the complete required timeframe for participants formed. The focus of this study was on characterizing to be on a stable dose of rhEPO or its analogs up to 4 weeks the hemoglobin-TIW dose-response relationship and prior to day − 28 through day 1. Those participants who using the results to estimate the dose conversion ratio were receiving maintenance oral or intravenous (IV) iron between once-daily and TIW doses that would produce supplementation were also required to be on a stable dose the same hemoglobin response, not on formal hypoth- from 4 weeks prior to day − 28 through day 29. esis testing. A sample size of approximately 90 random- Patients were ineligible for the study if they were be- ized participants was determined based on feasibility (18 ing treated with ≥360 IU/kg/week IV or ≥ 250 IU/kg/ per treatment group). week subcutaneous (SC) epoetin, ≥1.8 μg/kg/week IV The primary study endpoint was change in hemoglobin or SC darbepoetin, or ≥ 2.2 μg/kg/week methoxy poly- from baseline at day 29. Baseline hemoglobin was defined as ethylene glycol-epoetin beta within the 8 weeks prior the average of the hemoglobin measured at the day − 14 to randomization. Patients with low values of vitamin and day 1 visits. This change from the planned analysis, B12 (< 200 pg/mL [148 pmol/L]), folate (< 2.0 ng/mL which defined baseline hemoglobin as the day 1 hemoglobin [4.5 nmol/L]), ferritin (< 100 ng/mL [225 pmol/L]), or value, was made in order to reflect the overall pretreatment transferrin saturation (TSAT) (< 20%) were also ineli- stable hemoglobin value and was prespecified in the Report- gible. Additional exclusion criteria included a planned ing and Analysis Plan, which was finalized before database change in dialysis modality or renal transplant within freeze and unblinding. the study time period; significant cardiovascular, The dose-response relationship between daprodustat hematologic, hepatic, or chronic inflammatory disease; TIW and hemoglobin at day 29 was characterized using active gastrointestinal (GI) bleeding or GI bleeding a three-parameter Emax model fitted using Bayesian stat- within 8 weeks prior to day − 28 through day 1; QT istical methods. Emax was assumed to be an appropriate interval corrected for heart rate using Bazett’sformula model based on the biology of daprodustat and once- (QTcB) > 500 msec or QTcB > 530 msec in patients daily dose-response modeling completed to date for with bundle branch block; and malignancy within 2 daprodustat. Prior distributions for each of the model years of screening, currently receiving treatment for parameters (E0, Emax, ED50, and variance) used in this Bailey et al. BMC Nephrology (2019) 20:372 Page 4 of 12

trial were informed by the once-daily dose-response hemoglobin (CHr), which were summarized by visit (day from a previous phase 2 study (ClinicalTrials.gov identi- 1, 15, and 29) and treatment group. Additionally, max- fier NCT01977482) [9] This modeling provided confi- imum observed change from baseline in plasma EPO dence in setting prior distributions for E0, however, and maximum observed percent change from baseline in there was greater uncertainty in the estimation of Emax VEGF were assessed and summarized by treatment and ED50, which was explored in sensitivity analyses group. Descriptive PK summaries of daprodustat and with alternative (weaker) prior distributions, including a major metabolites were also summarized by daprodustat Bayesian three-parameter Emax model based on the com- dose level. In general, secondary analyses were descrip- pleter population, Bayesian three-parameter Emax model tive and estimation based. using sensitivity (weaker, less informative) priors, and Safety assessments included incidence and severity of linear dose-response model based on the intention-to- AEs and serious AEs (SAEs), reasons for discontinuation treat (ITT) population. of investigational product, discontinuations for safety- Once the dose-response model was fitted satisfactorily, related reasons, and absolute values and changes from the posterior distribution of the model parameters (E0, baseline over time in laboratory parameters, electrocar- Emax, ED50, and variance) was summarized through diograms (ECGs), and vital signs. Dose-dependent AE means, standard deviation (SD), median, Monte Carlo relationship was not assessed in this study. The investi- standard error (MCSE), MCSE/SD and equal-sided 95% gator or site staff was responsible for detecting, docu- credibility intervals. The predicted value of hemoglobin menting, and reporting AEs/SAEs. AEs were collected change from baseline at day 29 at a series of doses from the start of study treatment (day 1) and until the (range of possible doses from 0 to 50 was sectioned off follow-up visit. SAEs were recorded from the time of into a sequence of 0.5 increments; ie, dose levels of 0, subject consent to participate in the study up to and in- 0.5, 1.0, 1.5, 2.0….50) was also monitored and the me- cluding any follow-up contact. dian and 95% credibility values obtained at each dose. The observed change in hemoglobin from baseline at day 29 was used as the response variable in the Emax Results model. Participants who had a day 15 hemoglobin meas- Patient population urement, but a missing day 29 hemoglobin measure- Of 211 patients screened, 103 (49%) met the eligibility ment, were included with a day 29 value imputed using criteria and were randomized to the placebo group (n = 2 × (day 15 hemoglobin – baseline hemoglobin). Partici- 20) or one of the four daprodustat cohorts (n =83 in pants who prematurely discontinued dosing prior to day total) (Fig. 1). All 103 patients were included in the 15 were nonevaluable for the dose-response analysis. safety and PK populations and 97 were included in the The dose conversion ratio of the once-daily dose to ITT population (18 in the placebo group and 79 in the the corresponding TIW dose that produced an equiva- total daprodustat group). One participant randomized to lent hemoglobin response was estimated based on the the placebo group erroneously received daprodustat 25 dose-hemoglobin response relationship for the once- mg. This participant was counted in the placebo group daily regimen derived from a previous study [9] and for in the ITT population and in the daprodustat 25 mg co- the TIW regimen derived from the current trial. TIW hort in the safety and PK populations. Of all patients dosing and once-daily dosing were both estimated by randomized, 17 participants in the placebo group (85%) Bayesian Emax model. and 67 in the total daprodustat group (81%) completed The observed change in hemoglobin from baseline to the treatment period. With the exception of 1 partici- day 29 was analyzed by a repeated measures model using pant in the total daprodustat group, all patients who the PROC MIXED procedure. Model effects included completed the study also completed the follow-up visit. baseline hemoglobin, treatment, time, and treatment-by- The most common reason for study treatment dis- time interactions. The variance-covariance structures for continuation in the total daprodustat group was due repeated measures within the same subject were un- to participants reaching protocol-defined hemoglobin structured. For day 29, least squares mean differences stopping criteria. No participant receiving placebo dis- were calculated as estimates of the difference between continued study treatment due to meeting protocol- the daprodustat cohorts and placebo, together with 95% defined hemoglobin stopping criteria. confidence intervals (CIs). Demographic and baseline characteristics were gener- Additional secondary endpoints included observed ally balanced between the placebo and total daprodustat change from baseline in hepcidin, ferritin, transferrin, group, with the exception that the total daprodustat total iron, unsaturated iron binding capacity (UIBC), group contained more males (Table 1). The majority of total iron binding capacity (TIBC), hematocrit, red blood participants in both the placebo and total daprodustat cell (RBC) count, reticulocyte count, and reticulocyte group were white, the mean baseline hemoglobin level Bailey et al. BMC Nephrology (2019) 20:372 Page 5 of 12

Fig. 1 Study Flow Diagram. AE, adverse event; ITT, intention-to-treat; Hgb, hemoglobin; PK, pharmacokinetics; SAE, serious adverse event; TIW, three times weekly. aOne participant randomized to the placebo group erroneously received daprodustat 25 mg. This participant was counted in the placebo group in the ITT population and in the daprodustat 25 mg cohort in the safety and PK populations. bThe ITT population comprised all randomized participants who received at least one dose of study treatment, and had a baseline and at least one corresponding on-treatment assessment including hemoglobin was 10.6 g/dL, and approximately half of the participants hemoglobin remained near baseline levels in the 10 mg in both groups had diabetes. and 15 mg cohorts, and decreased in the placebo group. The model-adjusted treatment difference from placebo Change in hemoglobin from baseline to day 29 and over demonstrated that the treatment effect on hemoglobin time generally increased with the dose levels of daprodustat Switching from rhEPO to daprodustat produced dose- (Additional file 1: Table S2). dependent mean changes in hemoglobin (g/dL) from baseline to day 29 (Fig. 2 and Additional file 1: Table Dose-response model and dose conversion S1). Mean hemoglobin increased over time in the The dose-response relationship was well characterized daprodustat 25 mg and 30 mg cohorts, while mean within the dose range evaluated in this study (0–30 mg Bailey et al. BMC Nephrology (2019) 20:372 Page 6 of 12

Table 1 Demographic and Baseline Characteristics (ITT Population) Placebo Total Daprodustat Dose n =18 Daprodustat 10 mg 15 mg 25 mg 30 mg n =79 n =20 n =18 n =21 n =20 Age (years) Mean ± SD 60.9 ± 13.3 64.1 ± 15.0 63.6 ± 17.0 59.1 ± 12.7 67.2 ± 15.2 65.9 ± 14.3 Sex, n (%) Male 7 (39) 50 (63) 12 (60) 13 (72) 12 (57) 13 (65) Race, n (%) White 12 (67) 53 (67) 12 (60) 11 (61) 15 (71) 15 (75) African American 6 (33) 21 (27) 6 (30) 6 (33) 5 (24) 4 (20) Asian 0 2 (3) 1 (5) 1 (6) 0 0 Other 0 3 (4) 1 (5) 0 1 (5) 1 (5) Weight (kg) Mean ± SD 82.8 ± 26.5 78.8 ± 22.5 79.8 ± 26.0 79.8 ± 22.3 79.1 ± 24.3 76.3 ± 18.1 BMI (kg/m2) Mean ± SD 30.6 ± 8.6 28.2 ± 7.5 28.7 ± 8.6 27.4 ± 7.8 29.4 ± 7.4 27.1 ± 6.4 Diabetes, n (%) Yes 9 (50) 35 (44) 6 (30) 8 (44) 9 (43) 12 (60) Hgb (g/dL) Mean ± SD 10.6 ± 0.8 10.6 ± 0.7 10.6 ± 0.7 10.7 ± 0.8 10.4 ± 0.6 10.5 ± 0.7 Stratification factor, n (%) Low prior rhEPO dosea 14 (78) 59 (75) 15 (75) 14 (78) 16 (76) 14 (70) High prior rhEPO doseb 4 (22) 20 (25) 5 (25) 4 (22) 5 (24) 6 (30) Type of rhEPO, n (%) Darbepoetin 1 (6) 20 (25) 4 (20) 3 (17) 7 (33) 6 (30) Epoetin 17 (94) 59 (75) 16 (80) 15 (83) 14 (67) 14 (70) Standardized prior rhEPO dose (IU/kg/week) Mean ± SD 66.8 ± 43.1 86.6 ± 88.4 108.0 ± 147.1 75.0 ± 41.3 69.4 ± 51.2 93.7 ± 72.0 Smoking history,c n (%) N =19 N =84 N =20 N =20 N =22 N =22 Never 15 (79) 54 (64) 14 (70) 13 (65) 15 (68) 12 (55) Current 1 (5) 4 (5) 2 (10) 1 (5) 0 1 (5) Former 3 (16) 26 (31) 4 (20) 6 (30) 7 (32) 9 (41) Mode of dialysis,c n (%) N =19 N =84 N =20 N =20 N =22 N =22 Hemodiafiltration 3 (16) 18 (21) 3 (15) 3 (15) 6 (27) 6 (27) Hemodialysis 16 (84) 66 (79) 17 (85) 17 (85) 16 (73) 16 (73) BMI body mass index, Hgb hemoglobin, rhEPO recombinant human erythropoietin, ITT intention-to-treat, SD standard deviation aLow dose: < 100 IU/kg/week epoetin or < 0.5 μg/kg/week darbepoetin or < 0.6 μg/kg/week methoxy polyethylene glycol-epoetin beta bHigh dose: ≥100 IU/kg/week epoetin or ≥ 0.5 μg/kg/week darbepoetin or ≥ 0.6 μg/kg/week methoxy polyethylene glycol-epoetin beta cSafety population daprodustat administered TIW). There was high vari- conversion ratio between once-daily dosing and TIW dos- ability in individual participant’s hemoglobin response ing of daprodustat was estimated to be ~ 2.0 across the within treatment groups, and a large overlap in evaluated dose range when the estimation was based on hemoglobin response between adjacent doses (Fig. 3). the Bayesian three-parameter Emax model (Table 2). Overall, mean hemoglobin response increased with in- The target dose (dose expected to cause a 0 g/dL creasing daprodustat dose. change in hemoglobin) estimated by the three-parameter The dose-response relationship for once-daily dosing of Bayesian Emax model was 10.48 mg daprodustat adminis- daprodustat has been previously described [9]. The dose tered TIW (Table 2). Bailey et al. BMC Nephrology (2019) 20:372 Page 7 of 12

Fig. 2 Mean Change from Baseline and 95% CI for Hemoglobin (g/dL) Over Time (ITT Population). BL, baseline; CI, confidence interval; Hgb, hemoglobin; ITT, intention-to-treat

Participants who reached the hemoglobin stopping and 16 daprodustat participants) experienced increases criteria that met the protocol-defined hemoglobin stopping cri- Ten participants were withdrawn from the study based teria. Most of these 17 participants had received either on HemoCue measured hemoglobin values during the 25 mg or 30 mg daprodustat (14 participants), with the study. In a retrospective analysis of central laboratory remaining 3 participants having been in the two lower hemoglobin values, 2 participants experienced decreases daprodustat dose cohorts. The majority (11/13) of par- that met protocol-defined hemoglobin stopping criteria ticipants with a ≥ 1 g/dL increase in hemoglobin over the (1 participant in 10 mg cohort and 1 in 15 mg cohort, previous 2 weeks, including 9 of 11 participants who re- but none in placebo). Overall, 17 participants (1 placebo ceived either 25 mg or 30 mg daprodustat, were in the participant who mistakenly received 25 mg daprodustat low prior rhEPO (and its analogs) dose stratum.

Fig. 3 Posterior Emax Dose-Response Curve for Hemoglobin (g/dL) Change from Baseline at Day 29 (ITT Population). The two dotted lines are 95% credible bands. Emax, maximal response; Hgb, hemoglobin; ITT, intention-to-treat Bailey et al. BMC Nephrology (2019) 20:372 Page 8 of 12

Table 2 Summary of Dose Ratio Between Three-Times Weekly Doses and Once-Daily Doses and Estimated Doses Corresponding to Specific Hemoglobin Response Levels (ITT Population) Estimated TIW 95% CI Selected Hemoglobin Response Estimated QD Doses (mg) 95% CI Dose Ratio 95% CI Doses (mg) (TIW/QD)a (Bootstrap) 2.77 0.00–5.82 a change of -0.5 g/dL (minimally effective doseb) 1.27 0.00–2.63 2.19 0.49–5.20 6.44 3.21–9.99 a change of -0.25 g/dL 3.16 1.58–4.42 2.04 0.95–4.54 10.48 6.98–15.01 a change of 0 g/dL (target dosec) 5.26 3.91–6.62 1.99 1.25–3.17 14.93 10.71–20.97 a change of 0.25 g/dL 7.58 6.18–9.45 1.97 1.33–2.92 19.85 14.59–27.96 a change of 0.5 g/dL 10.18 8.40–13.08 1.95 1.32–2.89 25.30 18.74–36.16 a change of 0.75 g/dL 13.09 10.68–17.60 1.93 1.27–2.92 31.38 23.27–45.95 a change of 1 g/dL 16.38 13.08–23.33 1.92 1.21–2.98 CI confidence interval, ITT intention-to-treat, QD once daily; TIW, three times weekly a Both the TIW doses and the QD doses were estimated by Bayesian Emax model bThe smallest dose that achieves a change of −0.5 g/dL in Hgb over 29 days after being switched from ESA cThe dose that achieves a 0 g/dL change in Hgb over 29 days after being switched from rhEPO or its analogs

EPO and VEGF in the placebo group had a mean decrease in hematocrit Median baseline concentrations of plasma EPO were simi- and RBCs. Mean baseline reticulocyte counts were also lar across treatment groups. There was a dose-dependent similar across treatment groups and daprodustat trended increase in the observed circulating EPO concentration toward dose-dependent mean changes from baseline. For following administration of daprodustat TIW (Fig. 4). Day the daprodustat 25 mg and 30 mg cohorts, the observed 29 pre-dose EPO levels were near or below baseline values increase was smaller at day 29 than at day 15. in participants receiving daprodustat, indicating no accumulation of EPO after daprodustat TIW treatment. Pharmacokinetic results Median baseline plasma VEGF concentrations were Daprodustat was observed to be readily absorbed with similar among the daprodustat cohorts, but somewhat a median time to maximum observed plasma concen- higher in the placebo group (Fig. 4). Circulating VEGF tration (t ) value of approximately 2.0 h (Additional concentrations were highly variable in all dose cohorts max file 1: Table S5). Systemic exposures (area under the at all time points. Day 29 pre-dose VEGF concentrations concentration-time curve from time 0 to time t in participants receiving daprodustat were near or below [AUC ]) increased with dose, and were approxi- baseline, indicating no accumulation of VEGF after ad- 0-t mately linear with dose across the dose range (ie, for a ministration of daprodustat TIW. 3-fold increase in dose from 10 mg to 30 mg, AUC increased 3.2-fold). Geometric mean maximum observed Markers of Iron metabolism and utilization plasma concentration (C )exposuresweresimilar Both geometric mean baseline hepcidin and mean base- max between the 10 mg and 15 mg doses and higher for the line ferritin values were similar across treatment groups 25 mg and 30 mg doses. High inter-participant vari- and each daprodustat dose cohort. Daprodustat adminis- ability was noted for each PK parameter, with calcu- tration led to dose-dependent decreases from baseline at lated % coefficient of variation (CV) values > 90%. day 29 in geometric mean hepcidin and in mean ferritin (Additional file 1: Figure S1). Mean baseline values for transferrin, TIBC, UIBC, and Safety analysis CHr were also similar across all treatment groups and Overall, 26% of participants in the placebo group and trended toward increases in a dose-dependent manner 33% in the combined daprodustat group experienced at during the study (Additional file 1: Table S3). Both mean least one on-treatment AE (Table 3). In both groups, the baseline total iron and mean baseline TSAT were similar majority of on-treatment AEs were mild or moderate in across treatment groups, and there were overall no clinic- intensity. There was no dose-dependent relationship be- ally relevant changes from baseline for either parameter. tween AEs and daprodustat. The most frequently reported on-treatment AE in the total daprodustat group Indices of hematopoiesis was hypotension (3 participants, 4%). Three on- Mean baseline hematocrit and RBC counts were similar treatment AEs assessed by the investigator to be related across all treatment groups. Daprodustat administration to daprodustat were reported in 2 participants: dizziness led to a dose-dependent mean change from baseline in and gastroesophageal reflux disease in 1 participant in these parameters (Additional file 1: Table S4). Participants the daprodustat 10 mg cohort, and depression in 1 Bailey et al. BMC Nephrology (2019) 20:372 Page 9 of 12

Fig. 4 EPO and VEGF Absolute Values and Change From Baseline by Visit and Sample Time (ITT Population; Post-Hoc Analyses). Each boxplot displays the distribution of the parameter indicated on the Y-axis label. The bottom and top edges of the box indicate the intra-quartile range (IQR); that is the range of values between the 25th and 75th percentiles. The line inside the box indicates the median value. The whiskers are drawn from the box to the most extreme point that is less than or equal to 1.5 times the IQR. The dots are outliers that are more extreme than the upper and lower fences (±1.5 IQR). EPO, erythropoietin; ITT, intention-to-treat; VEGF, vascular endothelial growth factor participant in the daprodustat 15 mg cohort. No No adverse trends were apparent following review of treatment-related AEs were reported in the placebo clinical laboratory values, vital sign values, and ECG group. values, and no deaths were reported in the randomized There was a similar proportion of on-treatment population. SAEs reported in the total daprodustat group (10%, 8 participants) and placebo group (11%, 2 participants) Discussion (Table 3). Three SAEs in the daprodustat group oc- This was a dose-ranging study evaluating TIW dosing of curred in more than 1 participant (unstable angina, daprodustat in patients with anemia of CKD on congestive cardiac failure,andfluidoverload).All hemodialysis in order to estimate the dose conversion events resolved within 1 week. None of the SAEs were ratio between once-daily and TIW administration of considered by the investigator to be related to study daprodustat and help inform TIW doses for future stud- treatment. ies. The enrolled study population was representative of Two participants were withdrawn from study treat- the real-world hemodialysis population, which includes ment due to an SAE; both participants had been ran- ~ 30% African Americans. The results showed a dose- domized to one of the daprodustat arms. One dependent increase in hemoglobin with TIW daprodu- participant in the 15 mg cohort experienced a hyperten- stat, and demonstrated the ability of TIW daprodustat sive crisis and 1 participant in the 30 mg cohort experi- doses between 10 mg and 30 mg to initially maintain enced unstable angina. Both events resolved in 2 days. hemoglobin levels in patients switched from rhEPO or Neither event was considered by the investigator to be its analogs. The dose conversion ratio for once-daily to related to study treatment. TIW daprodustat was estimated to be approximately 2.0 Bailey et al. BMC Nephrology (2019) 20:372 Page 10 of 12

Table 3 Adverse Events (Safety Population) Preferred Term, n (%) Placebo Total Daprodustat Dose n =19 Daprodustat 10 mg 15 mg 25 mg 30 mg n =84 n =20 n =20 n =22 n =22 On-Treatment Common AEs ≥ 2 Participantsa Any Event 5 (26) 28 (33) 9 (45) 6 (30) 7 (32) 6 (27) Hypotension 0 3 (4) 1 (5) 1 (5) 1 (5) 0 Angina unstable 0 2 (2) 1 (5) 0 0 1 (5) Cardiac failure congestive 0 2 (2) 2 (10) 0 0 0 Diarrhea 0 2 (2) 2 (10) 0 0 0 Fluid overload 0 2 (2) 0 0 1 (5) 1 (5) Procedural hypotension 0 2 (2) 1 (5) 0 1 (5) 0 On-Treatment Serious AEsb Any Event 2 (11) 8 (10) 3 (15) 2 (10) 1 (5) 2 (9) Angina unstable 0 2 (2) 1 (5) 0 0 1 (5)c Cardiac failure congestive 0 2 (2) 2 (10)d 000 Fluid overload 0 2 (2) 0 0 1 (5)d 1 (5) Hypertensive crisis 0 1 (1) 0 1 (5)c 00 Mesenteric artery stenosis 0 1 (1) 0 1 (5) 0 0 Postprocedural myocardial infarction 0 1 (1) 1 (5) 0 0 0 Arteriovenous fistula thrombosis 1 (5) 0 0 0 0 0 Orthostatic hypotension 1 (5)d 0 0 000 AE adverse event aCommon AE is defined as an AE with ≥2 participants in any treatment group or ≥ 2% (based on unrounded value) in the total daprodustat group bParticipants could have ≥1AE cStudy-withdrawn or drug-withdrawn dDrug-interrupted across the evaluated dose range. These data can be used A higher proportion of participants treated with to calculate the TIW doses for future phase 3 studies daprodustat permanently discontinued study treatment evaluating long-term safety and efficacy of daprodustat due to reaching protocol-defined hemoglobin stopping TIW in patients with CKD, which would allow for criteria. However, the majority of those participants with greater dosing flexibility and dosing aligned with a TIW hemoglobin increases were receiving lower levels of hemodialysis schedule. rhEPO or its analogs prior to entry into the study. Since this is the first clinical study examining the Therefore, it is not unexpected that in this randomized effects of TIW daprodustat in the target patient popu- dose-ranging trial, treatment with doses of daprodustat lation, it was important to determine the effects these higher than required to maintain hemoglobin resulted in higher doses of daprodustat may have on relevant PD hemoglobin elevations requiring stopping dosing. This is markers. The study results are consistent with the less likely to occur when the prior EPO dose is used to mechanism of action of daprodustat to stabilize HIF, determine the starting dose for daprodustat TIW on an and similar to what has been observed with daprodu- individual basis. stat once daily, including dose-dependent changes in Daprodustat TIW was generally well tolerated and raised plasma EPO levels, over the 29-day treatment period no new safety concerns at doses up to 30 mg. Furthermore, without subsequent accumulation even at doses that the AEs observed are those typically seen in hemodialysis- increased hemoglobin levels. In contrast, due to high dependent patients and occurred with a similar frequency variability, there was no consistent dose-dependent in the placebo group and daprodustat treatment group. relationship observed in plasma VEGF levels. Changes Limitations of this study are the small sample size observed in markers of iron metabolism and indices and limited treatment duration which are inherent of a of erythropoiesis were also consistent with what dose-ranging trial. Additional limitations include the would be expected with increased erythropoiesis, and use of imputed values in the analysis for several partici- were similar to the trends seen with daprodustat once pants who met the hemoglobin stopping criteria. Add- daily. itionally, although the baseline characteristics were Bailey et al. BMC Nephrology (2019) 20:372 Page 11 of 12

reflective of the hemodialysis population, there were Funding more males in the total daprodustat group than in the Funding for this study (NCT02689206 available from www.clinicaltrials.gov) was provided by GlaxoSmithKline (GSK). All authors were employees of GSK placebo group. at the time of the study and participated in the design of the study, collection, analysis, and interpretation of data and in writing the manuscript. Conclusions This study estimated the dose conversion ratio for once- Availability of data and materials Portions of the dataset(s) generated or analyzed during this study are daily to TIW daprodustat to be ~ 2.0 across the evaluated included in this published article and its supplemental information file. dose range. The study also demonstrated the efficacy and The datasets generated and/or analyzed during the current study will safety of daprodustat TIW. A dose-dependent increase in be available within 6 months of this publication, anonymized individual participant data, the annotated case report form, protocol, reporting hemoglobin levels was seen when daprodustat was adminis- and analysis plan, data set specifications, raw dataset, analysis-ready tered TIW. The hemoglobin effects were achieved without dataset, and clinical study report will be available for research accumulation of plasma EPO or VEGF and with a safety proposals approved by an independent review committee. Proposals should be submitted to www.clinicalstudydatarequest.com.Adata profile consistent with the hemodialysis patient population. access agreement will be required. These data support future longer-term clinical studies in patients on hemodialysis to further explore daprodustat Ethics approval and consent to participate TIW to treat anemia of CKD. The study protocol was reviewed and approved by a national, regional, or investigational center ethics committee or institutional review board (Additional file 2). The study was conducted according to the Additional files recommendations of Good Clinical Practice and the Declaration of Helsinki. All patients provided written informed consent prior to participating in the study. Additional file 1: Table S1. Hemoglobin Over Time and Change from Baseline (ITT Population). Table S2. Repeated Measure Analysis for Hemoglobin Change from Baseline at Day 29 (ITT Population). Figure S1. Consent for publication A) Geometric Mean Change from Baseline and 95% CI for Hepcidin Not applicable. (ITT Population) and B) Mean Change from Baseline and 95% CI for Ferritin (ITT Population). Table S3. Change from Baseline in Markers of Competing interests Iron Metabolism at Day 29 (ITT Population). Table S4. Change from CKB, ARC, CH, KMM, and VVP are employees of and hold equity stock in Baseline in Indices of Hematopoiesis (ITT Population). Table S5. Summary GlaxoSmithKline (GSK). SC is a former employee of GSK and holds equity of Plasma Daprodustat Pharmacokinetic Parameters Following Dose stock in the company. Administration of Daprodustat (PK Population). (PDF 339 kb) Author details Additional file 2: Participating research centers and IEC/IRB committees. 1 (PDF 94 kb) R&D, Clinical Sciences, GlaxoSmithKline, 1250 S. Collegeville Road, Mail Code UP 4200, Collegeville, PA 19426, USA. 2R&D, GlaxoSmithKline, 1250 S. Collegeville Road, Collegeville, PA 19426, USA. 3R&D, Clinical Statistics, Abbreviations GlaxoSmithKline, 1250 S. Collegeville Road, Collegeville, PA 19426, USA. 4R&D, AE: Adverse event; AUC0-∞: Area under the concentration-time curve from Clinical Pharmacology, Modeling and Simulation, GlaxoSmithKline, 1250 S. 5 time 0 to time infinity; AUC0-t: Area under the concentration-time curve from Collegeville Road, Collegeville, PA 19426, USA. R&D, Discovery Medicine, time 0 to time t; BL: Baseline; BMI: Body mass index; CHr: Reticulocyte GlaxoSmithKline, 1250 S. Collegeville Road, Collegeville, PA 19426, USA. hemoglobin; CI: Confidence interval; CKD: Chronic kidney disease; Cmax: Maximum observed plasma concentration; CV: Coefficient of variation; Received: 3 May 2019 Accepted: 29 August 2019 ECG: Electrocardiogram; EPO: Erythropoietin; GI: Gastrointestinal; Hgb: Hemoglobin; HIF: Hypoxia-inducible factor; ITT: Intention-to-treat; IV: Intravenous; MCSE: Monte Carlo standard error; PBO: Placebo; References PD: Pharmacodynamic; PHI: Prolyl hydroxylase inhibitor; PK: Pharmacokinetic; 1. Babitt JL, Lin HY. Mechanisms of anemia in CKD. J Am Soc Nephrol. ’ QD: Once daily; QTcB: QT interval corrected for heart rate using Bazett s 2012;23:1631–4. formula; RBC: Red blood cell; rhEPO: recombinant human erythropoietin; 2. Zumbrennen-Bullough K, Babitt JL. The iron cycle in chronic kidney disease SAE: Serious adverse event; SC: Subcutaneous; SD: Standard deviation; (CKD): from genetics and experimental models to CKD patients. Nephrol SRT: Safety Review Team; t1/2: half-life; TIBC: Total iron binding capacity; Dial Transplant. 2014;29:263–73. TIW: Three times weekly; tmax: time to maximum observed plasma 3. FDA Drug Safety Communication: Modified dosing recommendations to concentration; TSAT: Transferrin saturation; UIBC: Unsaturated iron binding improve the safe use of erythropoiesis-stimulating agents (ESAs) in chronic capacity; VEGF: Vascular endothelial growth factor kidney disease. Safety announcement issued June 24, 2011. http://www.fda. gov/Drugs/DrugSafety/ucm259639.htm#sa. Accessed 12 Mar 2019. Acknowledgements 4. Kidney disease: improving global outcomes (KDIGO) anemia work group. All listed authors meet the criteria for authorship set forth by the KDIGO clinical practice guideline for anemia in chronic kidney disease. International Committee for Medical Journal Editors. Medical editorial Kidney Int Suppl. 2012;2:279–335. support (Sarah Hummasti, PhD) and graphic services were provided by AOIC, 5. Agarwal R, Kusek JW, Pappas MK. A randomized trial of intravenous and oral LLC and were funded by GlaxoSmithKline. The development of the iron in chronic kidney disease. Kidney Int. 2015;88:905–14. manuscript followed the Consolidated Standards of Reporting Trials 6. Gupta N, Wish JB. Hypoxia-inducible factor prolyl hydroxylase inhibitors: a (CONSORT) checklist. potential new treatment for anemia in patients with CKD. Am J Kidney Dis. 2017;69:815–26. Authors’ contributions 7. Haase VH. Mechanisms of hypoxia responses in renal tissue. J Am Soc SC, ARC, CH, and KMM were responsible for the conception or the design of Nephrol. 2013;24:537–41. the study. CKB, SC, ARC, CH, KMM, and VVP were responsible for the data 8. Holdstock L, Meadowcroft AM, Maier R, Johnson BM, Jones D, Rastogi analysis or interpretation of the data. All authors provided critical review and A, et al. Four-week studies of oral hypoxia-inducible factor-prolyl final approval of the manuscript for publication and agree to take hydroxylase inhibitor GSK1278863 for treatment of anemia. J Am Soc responsibility for the content. Nephrol. 2016;27:1234–44. Bailey et al. BMC Nephrology (2019) 20:372 Page 12 of 12

9. Meadowcroft AM, Cizman B, Holdstock L, Biswas N, Johnson BM, Jones D, et al. Daprodustat for anemia: a 24-week, open-label, randomized controlled trial in participants on hemodialysis. Clin Kidney J. 2019;12:139–48. 10. Dialysis Outcomes and Practice Patterns Study Program: DOPPS Practice Monitor. August 2015. http://www.dopps.org/DPM. Accessed 12 Mar 2019. 11. McGowan T, Vaccaro NM, Beaver JS, Massarella J, Wolfson M. Pharmacokinetic and pharmacodynamic profiles of extended dosing of epoetin alfa in anemic patients who have chronic kidney disease and are not on dialysis. Clin J Am Soc Nephrol. 2008;3:1006–14.

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