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The Antagonist Lowers Residual Albuminuria in Patients with Type 2 Diabetic Nephropathy

† † † ‡ † Dick de Zeeuw,* Blai Coll, Dennis Andress, John J. Brennan, Hui Tang, Mark Houser, | Ricardo Correa-Rotter,§ Donald Kohan, Hiddo J. Lambers Heerspink,* Hirofumi Makino,¶ †† ‡‡ || Vlado Perkovic,** Yili Pritchett, Giuseppe Remuzzi, Sheldon W. Tobe,§§ Robert Toto, Giancarlo Viberti,¶¶ and Hans-Henrik Parving***

*Department of Clinical Pharmacology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands; †Renal Clinical Development and ‡Data and Statistical Sciences, AbbVie, North Chicago, Illinois; §Salvador Zubiran National Medical Science and Nutrition Institute, Mexico City, Mexico; |Division of Nephrology, University of Utah Health Sciences Center, Salt Lake City, Utah; ¶Okayama University Graduate School of Medicine, Okayama, Japan; **George Institute for Global Health, University of Sydney, Sydney, Australia; ††Astellas Global Pharma Development, Inc., Northbrook, Illinois; ‡‡Azienda Ospedaliera Papa Giovanni XXIII and IRCCS-Instituto di Ricerche Farmacologiche Mario Negri, Bergamo, Italy; §§Sunnybrook Health Sciences Center, Toronto, Ontario, Canada; ||University of Texas Southwestern Medical Center, Dallas, Texas; ¶¶Kings’ College London School of Medicine, London, United Kingdom; and ***Department of Medical Endocrinology, Rigshospitalet University Hospital of Copenhagen, Copenhagen, Denmark

ABSTRACT Despite optimal treatment, including renin-angiotensin system (RAS) inhibitors, patients with type 2 diabetic nephropathy have high cardiorenal morbidity and mortality related to residual albuminuria. We evaluated whether or not atrasentan, a selective endothelin A , further reduces albuminuria when administered concomitantly with maximum tolerated labeled doses of RAS inhibitors. We enrolled 211 patients with type 2 diabetes, urine albumin/creatinine ratios of 300–3500 mg/g, and eGFRs of 30–75 ml/min per 1.73 m2 in two identically designed, parallel, multinational, double-blind studies. Participants were randomized to placebo (n=50) or to 0.75 mg/d (n=78) or 1.25 mg/d (n=83) atrasentan for 12 weeks. Compared with placebo, 0.75 mg and 1.25 mg atrasentan reduced urine albu- min/creatinine ratios by an average of 35% and 38% (95% confidence intervals of 24 to 45 and 28 to 47, respectively) and reduced albuminuria$30% in 51% and 55% of participants, respectively. eGFR and office BP measurements did not change, whereas 24-hour systolic and diastolic BP, LDL cholesterol, and triglyceride levels decreased significantly in both treatment groups. Use of atrasentan was associated with a significant increase in weight and a reduction in hemoglobin, but rates of peripheral , heart failure, or other side effects did not differ between groups. However, more patients treated with 1.25 mg/d atrasentan discontinued due to adverse events. After stopping atrasentan for 30 days, measured parameters returned to pretreatment levels. In conclusion, atrasentan reduced albuminuria and improved BP and lipid spectrum with manageable fluid overload–related adverse events in patients with type 2 di- abetic nephropathy receiving RAS inhibitors.

J Am Soc Nephrol 25: ccc–ccc, 2014. doi: 10.1681/ASN.2013080830

Received August 5, 2013. Accepted October 10, 2013. Correspondence: Dr. Dick de Zeeuw, Department of Clinical Pharmacology, University Medical Center Groningen, Sector F, Published online ahead of print. Publication date available at PO Box 196, 9700 AD Groningen, The Netherlands. Email: d.de. www.jasn.org. [email protected]

Copyright © 2014 by the American Society of Nephrology

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Despite an armamentarium of different intervention strate- Helicobacter pylori infection, n=1; acute appendicitis, n=1; gies, patients with type 2 diabetic nephropathy still have a high and lung hemorrhage, n=1). In addition, 12 patients (15%) risk for comorbid events, such as cardiovascular and renal receiving 1.25 mg/d atrasentan discontinued due to AEs, morbidity and mortality. In particular, renin-angiotensin mostly related to fluid overload (edema, n=6; anemia, n=2). system (RAS) inhibitors, including angiotensin-converting Other reasons for discontinuation included fatigue (n=2), enzyme (ACE) inhibitors or angiotensin receptor blockers hyperkalemia (n=1), hypoglycemia (n=1), coronary artery (ARBs), have been effective in reducing cardiovascular and stenosis (n=1), and thyroid cancer (n=1). Seven patients renal risk. However, despite optimal treatment according to changed their dose or prescription of RAS inhibitors (placebo, guidelines used for metabolic control, blood pressure control, n=2; 0.75 mg/d, n=1; and 1.25 mg/d, n=4) and one patient dis- and lipid management; patients with diabetic nephropathy still continued because of hyperkalemia at the lowest dose of RAS have a very high renal and cardiovascular risk.1 inhibitors. Overall, 13 patients changed the dose of RAS inhibitors have renal and cardiovascular protective (2 [4%], 4 [5.1%], and 7 [8.4%] participants in the placebo, properties beyond their BP-lowering capacity, which has been 0.75 mg/d, and 1.25 mg/d groups, respectively). Edema was partly attributed to their albuminuria-lowering effect. Indeed, the most common reason for increasing the dose in several post hoc analyses show that the albuminuria-lowering the 1.25 mg/d atrasentan group (n=4), compared with the pla- effect of RAS inhibitors is related to renal and cardiovascular cebo group (n=1) and the 0.75 mg/d group (n=1). protection.2–6 However, the high residual risk in diabetic nephrop- athy is related to the residual albuminuria in these patients.2 Patient Characteristics Novel treatment options are needed, especially drugs that The baseline demographics, clinical and biochemical charac- lower residual risk factors without increasing adverse events teristics, and concomitant medications were similar between (AEs). Selective endothelin A (ETA) receptor antagonists are a the three groups (Table 2). promising class of drugs that have been shown to lower albuminuria in patients with diabetic nephropathy.7 However, Primary Endpoint they also have some potentially limiting side effects, such as Repeated-measures analysis showed a significant decrease in fluid retention, with an increased risk for heart failure in pa- albuminuria for the 0.75 mg/d atrasentan (235.5% average tients with type 2 diabetes with nephropathy.8 Atrasentan is a reduction over 12 weeks) and 1.25 mg/d atrasentan (238.6% highly selective ETA receptor antagonist that has been shown average reduction over 12 weeks) groups compared with the to lower albuminuria with renoprotective properties.9 placebo group. Figure 2A shows the geometric mean change in In this study, we tested the efficacy and safety of two low the urinary albumin/creatinine ratio (UACR) from baseline to doses of atrasentan (0.75 and 1.25 mg/d) on albuminuria and each postbaseline visit. Patients receiving 0.75 mg/d atrasentan other renal risk–related parameters in pa- tients with diabetic nephropathy who were concomitantly treated with stable RAS in- hibitor therapy, and particularly evaluated the balance between albuminuria-lowering effects and fluid retention side effects.

RESULTS

Patient Disposition Figure 1 shows the disposition of patients. Of the 831 individuals screened, 212 were eligible for randomization and 211 re- ceived the study drug (placebo, n=50; 0.75 mg/d atrasentan, n=78; and 1.25 mg/d atrasentan, n=83). During the 12-week study, 28 participants (13.3%) discontinued (placebo, n=2; 0.75 mg/d, n=11; and 1.25 mg/d, n=15). As shown in Table 1, the most common reason for dis- continuation was for AEs in 18 participants (8.5%). Six patients (8%) receiving 0.75 mg/d atrasentan discontinued due to AEs Figure 1. Consolidated Standards of Reporting Trials diagram. This is a summary of the (peripheral edema, n=2; fatigue, n=1; disposition of study participants.

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Table 1. Summary of incidence of AEs Atrasentan Adverse Event Placebo (n=50) 0.75 mg/d (n=78) 1.25 mg/d (n=83) Any adverse event 34 (68) 55 (71) 61 (74) Any serious adverse event 4 (8) 7 (9) 6 (7) Peripheral edema 21 (42) 27 (35) 35 (42) Facial edema ——3(3.6) Congestive heart failure 1 (2.0) 1 (1.3) — Weight increase 1 (2.0) — 3(3.6) Headache 2 (4.0) 1 (1.3) 5 (6.0) Nasal congestion and rhinitis 2 (4.0) — 1(1.2) Diarrhea and constipation 7 (14) 10 (13) 17 (21) Deaths —— — Adverse event leading to discontinuation — 6 (8) 12 (15) of study drug Peripheral edema — 2 (2.6) 4 (4.8) Facial edema ——2(2.4) Anemia ——2(2.4) Fatigue — 1 (1.3) 2 (2.4) Hyperkalemia ——1(1.2) H. pylori infection — 1(1.3) — Hypoglycemia ——1(1.2) Coronary artery stenosis ——1(1.2) Acute appendicitis — 1(1.3) — Lung hemorrhage — 1(1.3) — Thyroid cancer ——1(1.2) Data are presented as n (%). had an absolute median UACR of 8786908 mg/g at baseline, compared with placebo for the proportion of patients that which was reduced to 5736787 mg/g (234.7%) after 2 weeks achieved $40% and $50% reductions in UACR (36.8% and of treatment, and remained stable ending at 5216816 mg/g 27.6% for the 0.75 mg/d group versus 42.3% and 34.6% for (235.8%) at 12 weeks (P,0.001 for both). In patients treated 1.25 mg/d group, respectively; P,0.001 versus placebo). with 1.25 mg/d atrasentan, the UACR decreased from The response to atrasentan was tested for the different 8266690 mg/g at baseline to 5156472 mg/g (240.7%) at baseline parameters. In particular, change from baseline to 2 weeks and 4706547 mg/g (243.6%) at week 12 (P,0.001 final on-treatment UACR was not significantly influenced by for both). However, for participants receiving placebo, base- whether baseline UACR was #1000 mg/g (239% and 246%) line UACR was 6716941 mg/g, and it was not significantly or .1000 mg/g (232% and 241%) for the 0.75 and 1.25 mg/d changed over time (Table 3). groups, respectively. Similarly, baseline differences in age (,65 At the end of the 30-day follow-up period without study versus $65 years), sex, race (white versus nonwhite), or coun- medication, the median UACRs were 7376912 mg/g, try (United States versus non–United States) did not signifi- 105161206 mg/g, and 7276821 mg/g for the placebo, cantly influence the effect of atrasentan on UACR reduction. 0.75 mg/d, and 1.25 mg/d groups, respectively. The geometric mean changes from baseline to recovery were 25.3%, +4.3%, Renal Function and 27.8% for the placebo, 0.75 mg/d, and 1.25 mg/d groups, Serum creatinine showed no significant change in any of the respectively, thus showing that UACR values returned to baseline three groups. Baseline eGFR was in the low to normal ranges values. (49.3613.3, 47.9614.6, and 50.1613.6 ml/min per 1.73 m2 for the placebo, 0.75 mg/d atrasentan, and 1.25 mg/d atrasentan Secondary Endpoints arms, respectively). For the 0.75 mg/d atrasentan group, the Albuminuria placebo-corrected changes in eGFR (in milliliters per minute The geometric mean change from baseline to final UACR was per 1.73 m2) were 0.44 at 2 weeks (P=0.35), 20.99 at 6 weeks significantly changed in the 0.75 mg/d (236.2%) and 1.25 mg/d (P=0.77), 21.08 at 12 weeks (P=0.79), and 1.16 at recovery (243.9%) groups compared with the placebo group (+2.0%; (P=0.22). For the 1.25 mg/d atrasentan group, the placebo- P,0.001 for both). In the 0.75 and 1.25 mg/d groups, 51.3% corrected changes in eGFR (in milliliters per minute per 1.73 m2) and 55.1%, respectively, of participants achieved a $30% reduc- were 20.79 at 2 weeks (P=0.75), 20.93 at 6 weeks (P=0.75), tion in UACR, respectively (P,0.001 versus placebo) (Figure 2B). 22.05 at 12 weeks (P=0.94), and 0.63 for recovery (P=0.34) The differences were also statistically significantly different (Supplemental Figure 1).

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Table 2. Demographics and baseline characteristics of the intent-to-treat population Atrasentan Characteristic Placebo (n=50) 0.75 mg/d (n=78) 1.25 mg/d (n=83) Age, yr 64.3 (9.0) 65.0 (9.8) 64.5 (8.8) Sex, n (%) Male 40 (80) 63 (81) 57 (69) Female 10 (20) 15 (19) 26 (31) Race, n (%) White 23 (46) 36 (46) 38 (46) Black 2 (4) 14 (18) 13 (16) Asian 24 (48) 25 (32) 28 (34) Other 1 (2) 3 (4) 4 (5) Ethnicity, n (%) Hispanic or Latino 30 (60) 36 (46) 42 (51) Other 20 (40) 42 (54) 41 (49) Weight, kg 84.3 (20.2) 87.1 (22.1) 88.3 (18.4) Known duration of diabetes, yr 14.5 (9.5) 15.3 (9.3) 16.9 (9.4) BP, mmHg SBP 136 (14) 138 (14) 136 (15) DBP 72 (10) 75 (10) 74 (9) Serum albumin, g/L 40.1 (4.2) 40.3 (3.7) 40.5 (3.2) Serum creatinine, mg/dl 1.50 (0.38) 1.60 (0.44) 1.40 (0.35) eGFR, ml/min per 1.73 m2 49.3 (13.3) 47.9 (14.6) 50.6 (13.6) Hemoglobin, g/L 12.7 (1.8) 12.9 (1.5) 12.9 (1.8) Hemoglobin A1c, % 7.4 (1.3) 7.5 (1.5) 7.7 (1.4) Cholesterol, mg/dl Total 182 (48) 172 (42) 172 (39) LDL 100 (40) 91 (34) 88 (30) HDL 47 (12) 46 (14) 45 (12) Triglycerides, mg/dl 165 (83) 182 (129) 193 (112) Serum potassium, mmol/L 4.62 (0.49) 4.54 (0.53) 4.50 (0.51) UACR, median (Q1 to Q3), mg/g creatinine 671 (410–1536) 878 (515–1682) 826 (481–1389) Antihypertensives, n (%) RAS inhibitors 50 (100) 78 (100) 83 (100) b-Blockers 16 (32) 31 (40) 38 (46) Calcium channel blockers 27 (54) 42 (54) 46 (55) Diuretics, n (%) Loop diuretics 19 (38) 29 (37) 27 (33) 29 (58) 42 (54) 43 (52) Glucose-lowering therapies, n (%) glargine 12 (24) 25 (32) 23 (28) Metformin 13 (26) 19 (24) 22 (27) Sulphonylurea 27 (54) 33 (42) 32 (39) Statins, n (%) 38 (76) 58 (74) 68 (82) Coronary artery disease, n (%) 8 (16) 13 (16) 9 (10) Stroke, n (%) 10 (20) 8 (10) 8 (9) Data are presented as the mean (SD) unless otherwise noted.

Safety Results DBP was significantly changed in both groups compared with BP placebo (24.2 and 24.6 mmHg for 0.75 and 1.25 mg/d atrasentan After an initial reduction, overall office systolic BP (SBP) was not groups, respectively; P,0.001 for both). significantly different from baseline in either atrasentan group (Figure3A).However,overalldiastolicBP(DBP)wassignificantly Serum Lipids changed (21.9 mmHg for 0.75 mg/d, P=0.07; and 22.7 mmHg Intriguingly, atrasentan lowered mean total cholesterol, LDL for 1.25 mg/d, P=0.01) compared with placebo. cholesterol, and triglycerides over 12 weeks of treatment In contrast, 24-hour ambulatory SBP fell significantly for the (Figure4,A,B,andD).Totalcholesterolwassignificantly 0.75 mg/d (24.5 mmHg, P=0.03) and 1.25 mg/d (25.4 mmHg, lowered by 16.8 and 18.6 mg/dl for the 0.75 and 1.25 mg/d P=0.01) atrasentan groups (Figure 3B). Similarly, overall 24-hour atrasentan groups, respectively, compared with placebo

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We tested whether the multiple effects of atrasentan on albuminuria, BP, and lipids could be related to each other. Only a minor correlation was found between changes in SBP and changes in albuminuria for the 0.75 mg/d atrasentan group (SBP: r=0.24, P=0.04; DBP: r=0.05; P=0.66) and the 1.25 mg/d atrasentan group (SBP: r=0.30, P=0.01; DBP: r=0.24, P=0.03). Similarly, the correlation between albuminuria changes and lipid changes were minimal for the 0.75 mg/d atrasentan group (r=0.25, P=0.04) and absent for the 1.25 mg/d atrasentan group (r=0.01, P=0.95). Figure 2. Atrasentan treatment significantly decreases albuminuria. (A) UACR change in the percent geometric mean from baseline to recovery for the placebo (●), 0.75 mg/d AEs ▪ ▲ atrasentan ( ), and 1.25 mg/d atrasentan ( ) groups. (B) Degree of UACR reduction Overall mean change in body weight did not fi from baseline to nal in the placebo (squares), 0.75 mg/d atrasentan (open bars), and change significantly after low-dose (0.75 fi P, 1.25 mg/d atrasentan ( lled bars) groups. *** 0.001. mg/d) atrasentan treatment (0.4 kg; P=0.14 versus placebo), whereas high dose (1.25 mg/d) atrasentan significantly increased weight by 0.9 kg (P,0.001) compared with placebo over 12 weeks (Figure 5). An initial and significant increase in weight was observed for both doses of atrasentan compared with baseline (Figure 5). In pa- tients receiving 0.75 mg/d atrasentan, the differences were no longer significant at week 10, and weight returned to baseline values at week 12. However, weight did not return to baseline levels until 30 days post- treatment for participants receiving 1.25 mg/d atrasentan. Signs of hemodilution were observed in both atrasentan groups as indicated by a small but significant decrease in hemoglobin and hematocrit (Table 3). Figure 3. Atrasentan treatment modulates office and 24-hour ambulatory blood At baseline, the prevalence of edema was pressure. Mean office SBP/DBP over 12 weeks and after recovery (A) and mean 24-hour 47%, 31%, and 27% for the placebo, 0.75 ambulatory SBP/DBP over 6 and 10 weeks (B) for the placebo (circles), 0.75 mg/d atrasentan mg/d, and 1.25 mg/d groups. After 12 weeks P (squares), and 1.25 mg/d atrasentan (triangles) groups. Overall values are as follows: 0.63 of treatment, the overall rate of new or and 0.23 for 0.75 and 1.25 mg/d atrasentan, respectively, for SBP; 0.07 and 0.01 for worsening edema was comparable for the 0.75 and 1.25 mg/d atrasentan, respectively, for DBP; 0.03 and 0.01 for 0.75 and 1.25 mg/d atrasentan, respectively, for 24-hour SBP; and ,0.001 for 0.75 and 1.25 mg/d atrasentan, placebo (42%), 0.75 mg/d (35%), and 1.25 respectively, for 24-hour DBP. mg/d (42%) groups and was mostly mild in severity. Figure 5, bottom, shows the weekly incidence of new or worsening (P,0.001 for both versus placebo). LDL cholesterol decreased edema during the study. Moreover, the change in the dose of significantly by 14.6 mg/dl for both atrasentan doses compared diuretics was not different between treatment groups during with placebo (P,0.001 for both versus placebo). Similarly, 0.75 the study (4%, 5%, and 8% for the placebo, 0.75 mg/d, and mg/d atrasentan reduced triglycerides by 30.2 mg/dl (P=0.11), 1.25 mg/d groups, respectively). The mean durations of edema whereas 1.25 mg/d atrasentan significantly lowered triglycerides were 24.1, 29.5, and 33.8 days for the placebo, 0.75 mg/d, and by 47.9 mg/dl (P=0.01) compared with placebo; however, HDL 1.25 mg/d groups, respectively. cholesterol did not change (Figure 4C). Lipid profiles recovered Two patients developed congestive heart failure in the 30 days post-treatment with atrasentan and similar lipid-lowering placebo and 0.75 mg/d groups (n=1 each) (Table 1). The par- effects were observed in patients receiving statins versus those ticipant in the placebo group was diagnosed at week 12 with a patients not receiving statins (data not shown). previous event of edema at week 6, and the patient receiving

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0.75 mg/d atrasentan was diagnosed after an appendectomy at week 9 (Table 1). In addition, brain natriuretic levels did not change significantly over 12 weeks in the 0.75 mg/d (+1.069.2 pg/ml) and 1.25 mg/d (+18.869.0 pg/ml) groups com- pared with the placebo group (+29.5611.5 pg/ml). Table 1 shows a summary of AEs and AEs leading to discontinuation. Al- though it was not statistically significant, a higher number of patients (n=8) discontin- ued the study due to fluid retention–related events (edema, facial edema, and anemia) after treatment with the highest dose of atrasentan (1.25 mg/d) compared with the 0.75 mg/d dose (n=2) or placebo (n=0).

DISCUSSION

The low dose (0.75 mg/d) of the ETA receptor antagonist, atrasentan, lowered albuminuria by 36% without major side effects in patients with type 2 diabetes with nephropathy who were treated with the maximal tolerated labeled dose of ACE inhibitors or ARBs. Moreover, atrasentan had a mild BP-lowering effect and signifi- Figure 4. Atrasentan treatment exerts lipid-lowering effects. Mean changes in total cantly improved lipid profiles. Conversely, cholesterol (A), LDL cholesterol (B), HDL cholesterol (C), and triglycerides (D) after the higher dose of atrasentan (1.25 mg/d) placebo (●), 0.75 mg/d atrasentan (▪), and 1.25 mg/d atrasentan (▲) treatment for had a similar albuminuria-lowering effect 12 weeks and after recovery. but elicited more fluid retention. antagonists have been available since the early 1990s. Many preclinical studies with animal models have suggested that selective blockade of the ETA receptor is associated with renal protection when used in addition to independent proven therapies, such as RAS interventions.9 Although the mechanism of their re- noprotective effect is still debated, three important path- ways have clearly emerged. First, ETA receptor blockade has vascular effects, which causes glomerular vasodilation. This alters the glomerular permeability for albumin, thus lowering the tubular load of albumin.9 Both the hemody- namic and albuminuria-lowering effects of atrasentan have been associated with renal protection. Second, endothelin has been associated with (renal) inflammation,and,thus, ETA receptor blockade reduces renal inflammation by miti- gating the inflammatory effects of albuminuria reabsorp- tion and degradation in the proximal tubule and interstitium.10 Third, the endothelin system has been implicated in the depo- sition of and fibrosis,11 and, thus, ETA receptor antag- fi fi 7 Figure 5. Atrasentan treatment affects body weight. Mean changes in onists may be bene cial for reducing brosis in the . weight and new incidence of edema after placebo (●), 0.75 mg/d This study was not designed to explore the mechanism by atrasentan treatment (▪), and 1.25 mg/d atrasentan treatment (▲) which atrasentan lowers albuminuria. In addition, the current and after recovery. data did not resolve the question of whether the BP-lowering

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Table 3. Changes in main physical and biochemical parameters induced by placebo or atrasentan at different time points Atrasentan Placebo Variable Time (wk) 0.75 mg/d 1.25 mg/d n Mean (SD) or Median n Mean (SD) or Median n Mean (SD) or Median UACR, mg/g Baseline 50 671 78 878 83 826 2 48 696 75 573 82 515 6 48 686 74 636 75 461 12 48 797 70 521 69 470 Recovery 45 737 68 1051 71 727 SBP, mmHg Baseline 50 136 (14) 78 138 (14) 83 136 (15) 2 48 134 (16) 77 135 (16) 82 132 (16) 6 48 137 (12) 74 137 (17) 75 133 (17) 12 48 134 (16) 70 136 (15) 69 133 (18) Recovery 44 134 (13) 61 141 (19) 70 135 (16) DBP, mmHg Baseline 50 72 (10) 78 75 (10) 83 74 (9) 2 48 71 (9) 77 71 (11) 82 69 (10) 64871(10)7470(11)7569(9) 12 48 71 (10) 70 72 (9) 69 72 (12) Recovery 44 71 (10) 61 75 (11) 70 73 (11) 24-h SBP, mmHg Baseline 38 140 (14) 63 142 (15) 66 140 (14) 6 20 137 (14) 33 137 (16) 29 134 (16) 10 32 139 (16) 39 134 (15) 37 134 (15) 24-h DBP, mmHg Baseline 38 74 (9) 63 76 (9) 66 73 (8) 6 20 72 (8) 33 70 (8) 29 69 (8) 10 32 73 (8) 39 71 (9) 37 69 (9) LDL cholesterol, mg/dl Baseline 49 101 (39) 75 88 (34) 77 87 (30) 12 46 102 (38) 63 77 (30) 64 74 (26) Recovery 44 98 (39) 68 89 (36) 67 84 (31) eGFR, ml/min per 1.73 m2 Baseline 50 49 (13) 78 48 (15) 83 51 (14) 24848(14)7447(15)8349(14) 64848(15)7446(15)7449(15) 12 48 48 (14) 68 47 (16) 69 48 (15) Recovery 47 45 (13) 74 45 (17) 77 47 (14) HbA1C, % Baseline 50 7.4 (1.3) 78 7.5 (1.5) 83 7.7 (1.4) 12 48 7.3 (1.2) 69 7.1 (1.4) 75 7.2 (1.3) Hemoglobin, mg/dl Baseline 50 12.7 (1.8) 78 12.9 (1.5) 83 12.9 (1.8) 2 47 12.5 (1.9) 74 12.0 (1.5) 80 11.8 (1.8) 6 48 12.6 (1.9) 72 11.9 (1.7) 73 11.6 (1.7) 12 47 12.6 (2.1) 69 11.8 (1.6) 68 11.8 (1.8) Recovery 26 12.3 (1.9) 39 12.4 (1.5) 47 12.5 (1.9) Hematocrit, % Baseline 50 38.1 (5.4) 78 38.7 (4.4) 83 38.6 (5.3) 2 47 37.5 (5.8) 74 36.1 (4.6) 80 35.4 (5.2) 6 48 37.8 (5.5) 72 35.6 (5.0) 73 35.1 (5.2) 12 47 37.9 (6.3) 69 35.6 (4.8) 68 35.6 (5.5) Recovery 26 37.0 (6.2) 39 37.2 (4.5) 47 37.9 (5.7) Weight, kg Baseline 50 84.3 (20.2) 78 87.1 (22.1) 83 88.3 (18.4) 2 48 83.2 (19.6) 77 88.0 (22.2) 82 89.4 (18.7) 6 48 83.3 (19.6) 74 87.3 (21.8) 75 89.6 (19.0) 12 48 82.8 (18.6) 70 87.3 (22.3) 69 89.0 (19.2) Recovery 44 83 (20) 61 85 (23) 70 88 (19) effect of atrasentan is associated with a renal hemodynamic renoprotective profile. The clinical effects of endothelin effect that controls albuminuria. This study also did not resolve antagonists are similar to the preclinical: systemic and renal the question of whether the albuminuria-lowering effect is vasodilation and albuminuria-lowering effects. However, ob- linked to improvement of the lipid profile. servations from clinical studies indicate that these compounds One may wonder why endothelin antagonists have not yet also elicit volume-related side effects. This was particularly emerged in clinical practice, despite their clear, preclinical evident with the highest dose of avosentan, another ETA

J Am Soc Nephrol 25: ccc–ccc, 2014 Atrasentan Reduces Macroalbuminuria 7 CLINICAL RESEARCH www.jasn.org antagonist, during a large multidose phase IIa study in patients day has demonstrated the best efficacy (albuminuria-lowering) with diabetes.12 Despite this finding, a hard renal outcome and safety (AE profile) effect thus far. For this reason, the 0.75 study was started with high doses of avosentan, which was mg/d dose has been selected for future studies. prematurely terminated because of a 3-fold increase in heart Intriguingly, atrasentan also improved lipid profiles (total failure compared with placebo.8 This unfortunate outcome and LDL cholesterol). This study did not explore potential nearly ended the future of endothelin antagonists in the renal mechanisms mediatingthe lipid-lowering effects of atrasentan, arena. Besides the side effect of fluid retention, some endothelin which could be an indirect effect of the albuminuria-lowering receptor antagonists (especially those with a sulfonamide group) effect of atrasentan. Similar effects have been observed with have been linked to an increased risk of toxicity.13,14 Taken drugs that intervene in the RAS, which significantly reduce together, these studies show that endothelin antagonists have a proteinuria.18 narrow therapeutic window, and working within this window In this study, the efficacy of atrasentan was independent of may be the key to success. the baseline characteristics of the patients; in particular, neither An important observation that was drawn from past clinical baseline levels of albuminuria, nor age, sex, or geographic studies is that the dose response associated with fluid retention location determined the albuminuria-lowering response. This is different from the dose for albuminuria-lowering. One study was not powered to detect differences between partici- possibility is that fluid retention is driven by the endothelin B pating countries (e.g., Japan versus the United States and Canada); receptor blocking capacity of the drugs, although there are also however, when both studies were analyzed separately, we did not reports of sodium retention induced by ETA receptor blockade. find differences in key variables, such as albuminuria reduction or Even though most clinical endothelin receptor blockers are increase in weight (data not shown). In contrast, sitaxentan (an- developed with high ETA receptor selectivity, high doses of other endothelin receptor blocker) exerted a greater albuminuria- such drugs could still block the . If so, lowering effect in patients with high baseline albuminuria.19 lower doses of highly selective endothelin receptor antagonists However, whether this is due to differences in the two drugs or could be the way forward for effective clinical use of this drug due to the relatively small sample size of the sitaxentan study class, and atrasentan is an example of such a selective receptor remains unclear. blocker.15 Recent dose response studies by Kohan et al. showed It is noteworthy that this study was designed to address the that atrasentan has a remarkable capacity to lower albumin- effects of atrasentan in a patient population with the highest uria when used in addition to ACE inhibitor/ARB therapy unmet medical need. The enrolled patients had CKD stages 2 without overt signs of fluid retention at lower doses.16 How- and 3, all had macroalbuminuria and were receiving maximum ever, the sample size of the study was too small to draw any tolerated labeled doses of RAS inhibitors, and most of them final conclusion, which prompted this study. Indeed, we con- received diuretics. Despite these characteristics, albuminuria firm the very effective albuminuria-lowering capacity of the reduction was clinically highly relevant. Another strength of two atrasentan doses that were tested, and, importantly, the design of this study is that blood/urine was collected 30 days atrasentan did not cause a higher incidence of heart failure. after stopping therapy, and we observed that a majority of However, both atrasentan doses were associated with signs efficacy and safety variables returned to baseline, indicating the and/or symptoms of fluid overload. The highest dose (1.25 hemodynamic nature of the drug response. mg/d) promoted weight gain; thus, an optimal dose is critical What are the chances that 0.75 mg/d atrasentan per day to achieve the maximal albuminuria-lowering effect with will be renoprotective and delay hard endpoints? First, the minimal fluid retention. In addition, 12 patients receiving albuminuria-lowering response, both average as well as 1.25 mg/d atrasentan (15%) discontinued use due to AEs, proportion of responders, is exceptionally strong; thus far, more than half of which were related to fluid retention (8 there are no published reports of a drug added to RAS patients had either edema or anemia). Compared with placebo inhibitors that shows such a strong albuminuria-lowering (none), more patients receiving 0.75 mg/d atrasentan discon- effect. Second, the drug does not cause side effects, such as tinued (n=6, 8%), but only two patients discontinued due to hyperkalemia or hypotension, that have eliminated dual edema and the other causes were not likely linked to fluid RAS blockers. Third, it is unlikely that 0.75 mg/d atrasentan overload–related events. Even though weight increased in per day will cause major cardiovascular problems, as participants receiving 0.75 mg/d atrasentan in the first weeks observed in the study to Assess the Effect of the Endothelin of the study, it returned to baseline values by week 10, Receptor Antagonist Avosentan on Time to Doubling of without a significant increase in the use of diuretics (n=4, Serum Creatinine, End Stage Renal Disease or Death in 5.1%) compared with placebo (n=2, 4%). Lower doses of Patients With Type 2 Diabetes Mellitus and Diabetic atrasentan have been tested for efficacy and safety,16 and al- Nephropathy (ASCEND) clinical trial.8 Finally, the BP- and though the 0.25 mg/d dose was well tolerated, it showed a weak lipid-lowering effects of the drug may help reduce vascular albuminuria-lowering effect. In another phase IIb study,17 events. In that regard, a phase III clinical trial (Study of Diabetic 0.5 mg/d atrasentan did not significantly reduce albuminuria Nephropathy with Atrasentan [SONAR]; ClinicalTrials.gov compared with placebo (ClinicalTrials.gov identifier identifier NCT01858532) has just begun and will shed further NCT01399580). Taken together, 0.75 mg/d atrasentan per light on the renal effects of atrasentan.20

8 Journal of the American Society of Nephrology J Am Soc Nephrol 25: ccc–ccc,2014 www.jasn.org CLINICAL RESEARCH

In conclusion, 0.75 mg atrasentan per day markedly lowers Randomization and Blinding albuminuria in patients with type 2 diabetes with nephropathy Participants were asked to return three consecutive FMV urine with manageable fluid overload–related side effects. This pro- samples during the last run-in week and only those that met all of the file, combined with the lipid- and BP-lowering effects, appears inclusion criteria and none of the exclusion criteria were randomized. to make this drug an interesting candidate for clinical devel- For study 1, participants were randomized using an interactive voice opment to reduce the unmet need in diabetic renal disease response system with two stratification factors: country where the progression. participant was enrolled and the participant’s UACR level at week 1 (#1000 mg/g [113 mg/mmol], or .1000 mg/g [113 mg/mmol]). For study 2, participants were randomized using a Central Case Registra- CONCISE METHODS tion and Randomization Center using UACR level at week 1 (#1000 mg/g [113 mg/mmol], or .1000 mg/g [113 mg/mmol]) as a fi Study Design strati cation factor. Participants were assigned to placebo, 0.75 Data from two identically designed phase IIb, randomized, double- mg/d atrasentan, or 1.25 mg/d atrasentan packaged identically in a blind, parallel-designed, placebo-controlled, 12-week, multicenter 1:2:2 ratio in study 1 or in a 1:1:1 ratio in study 2. The study investi- ’ studies were pooled for analysis. Both studies were identical in patient gator, participant, and sponsor remained blinded to the participant s population, doses of atrasentan, and study procedures, but were treatment group assignment and UACR results for the duration of the conducted in different geographic regions (ClinicalTrials.gov iden- study. Participants were allowed to modify the dose of RAS inhibitors tifiers NCT01356849 for study 1 and NCT01424319 for study 2). The or diuretics during the study only if medically necessary. efficacy and safety of two doses of atrasentan were evaluated based on the reduction of albuminuria in patients with type 2 diabetes and Procedures nephropathy who were simultaneously receiving the maximum Study visits were scheduled for week 1 and week 2 and occurred every tolerated labeled daily dose of a RAS inhibitor, 90% of whom were also other week thereafter. Physical examination, including measurement receiving a diuretic. Patients were enrolled from 93 sites in the United of BP and weight, and assessment of edema were performed at every States, Canada, Taiwan (study 1), and Japan (study 2). After screening, visit. Full details on assessment of edema are provided in Supplemen- there was a run-in period of 4–12 weeks (4 weeks for patients already tal Methods. Supplemental Tables 3 and 4 show individual summaries receiving the maximum tolerated labeled dose of RAS inhibitors and a of adverse events for study 1 (NCT01356849) and study 2 diuretic; otherwise, participants were titrated up to the maximum (NCT01424319), respectively. Three FMVs were collected at each tolerated labeled dose for 4–12weeks).Eligiblepatientsthen visit, with the exception of week 1. In addition, 24-hour ambulatory entered a 12-week treatment period with a follow-up visit 4 weeks BP measurements were performed three times during the study: at after discontinuation of the study drug. Individual demographics and baseline and at weeks 6 and 10. Blood samples were collected under baseline characteristics results for study 1 (NCT01356849) and study 2 fasting conditions at baseline, week 12 (or premature discontinua- (NCT01424319) are shown in Supplemental Tables 1 and 2, respec- tion), and at the 30-day follow-up visit. tively. The study protocol was approved by an independent ethics committee and local and central review boards, and all participants Outcomes signed written informed consent before any study-specific procedures The primary efficacy endpoint was the change from baseline to week commenced. 12 in log-transformed UACR. Secondary efficacy measures included the change from baseline to final on-treatment log-transformed Eligibility Criteria and Recruitment UACR, the proportion of participants who achieved at least a 30%, At screening, eligible participants had type 2 diabetes with nephrop- 40%, and 50% reduction from baseline to final on-treatment UACR athy, were receiving hypoglycemic medication and a RAS inhibitor level, and the mean change in eGFR from baseline to each postbaseline within the previous 12 months, and had a UACR$300 and #3500 mg/g measurement (up to week 12). The same endpoints were used to calculated from two first morning void (FMV) urine samples, eGFR of evaluate participants at 30 days after stopping treatment. 30–75 ml/min per 1.73 m2 by Chronic Kidney Disease Epidemiology Collaboration formula, brain concentration,200 Statistical Analyses pg/ml, hemoglobin.9 g/dl, and serum albumin.3 g/dl. At the end of The planned sample size was 30 for the placebo group and 60 for each the run-in period, participants were eligible if their SBP was stable atrasentan dose group for study 1 and 18 per group for study 2. This (110–160 mmHg), serum potassium was ,5.5 mEq/L, and UACR was sample size would have provided at least 85% power to detect, at a one- .200 mg/g. Women were nonpregnant and postmenopausal for at sided level of significance of 0.05, a treatment group difference of least 1 year or were surgically sterile. Participants were excluded from 0.44 in the change from baseline to week 12 in log-transformed UACR the trial based the following criteria: a history of moderate or severe between each dose group and placebo (assuming a SD of 0.67) for edema, pulmonary edema, , heart failure, re- study 1, and at least 80% power to detect the overall treatment group ceiving loop diuretic therapy (.120 mg/d furosemide, .3mg/d difference in log-transformed UACR change from baseline over 12 bumetanide, .150 mg/d ethacrynic acid, or .60 mg/d torasemide), weeks of treatment between each dose group and placebo with two- and recent coronary artery disease. Full inclusion and exclusion sided significance level of 0.05 for study 2. All efficacy and safety criteria are provided in the Supplemental Methods. analyses were conducted on the data obtained from the intent-to-treat

J Am Soc Nephrol 25: ccc–ccc, 2014 Atrasentan Reduces Macroalbuminuria 9 CLINICAL RESEARCH www.jasn.org population, which consisted of data from all randomized participants DISCLOSURES whoreceivedatleastonedoseofstudydrug.Analyseswerebasedon D.D.Z. is a consultant for and receives honoraria (to employer) from AbbVie, comparisons between placebo and atrasentan groups as assigned by Astellas, AstraZeneca, Chemocentryx, Johnson & Johnson, Hemocue, Novartis, randomization, regardless of whether participants followed Reata Pharmaceuticals, Takeda, and Vitae. R.C.-R. has consultancy agree- through the protocol or were fully compliant with the protocol ments with Roche, AbbVie, Amgen, Fresenius Medical Care, and Boehringer; procedures. The primary analysis was a mixed-effects model in the last 2 years, he has been in the speakers’ bureau of Abbott, Amgen, Roche, fi repeated-measures analysis of change from baseline to each post- and Sano . D.K. is a consultant for AbbVie, Bristol-Myers Squibb, Reata baseline measurement of log UACR. The model included the fixed Pharmaceuticals, and Retrophin. H.J.L.H. is a consultant for and receives honoraria (to employer) from AbbVie, Astellas, Johnson & Johnson, Reata effects of treatment, country (study 1 only), visit, and treatment-by- Pharmaceuticals, and Vitae. H.M. is a consultant for AbbVie and Astellas; visit interaction, with covariates of baseline measurements and receives speaker honoraria from Astellas, MSD, Takeda, and Tanabe-Mitsubishi; baseline-by-visit interaction. The contrasts between each atrasentan and receives grant support from Astellas, MSD, Daiichi Sankyo, Dainippon dose group and the placebo group at week 12 were compared using a Sumitomo, Takeda, and Novo Nordisk. V.P.is a consultant for AbbVie, Astellas, one-sided significance level of 0.05; treatment differences at other AstraZeneca, Boehringer Ingelheim, Janssen, Roche, Servier, Merck, and Vitae; time points were also analyzed. The within-group geometric mean his employer receives funding/contracts for clinical trials from AbbVie, Baxter, change (%) is derived by 1003(exp(LS mean change)21), and the Fresenius, Novartis, Pfizer, Resmed, Roche, Janssen, and Servier. Y.P.is a former same transformation is applied on the 95% confidence interval employee of Abbott and owns AbbVie stocks. G.R. is a consultant for Alexion limits to obtain an approximate 95% confidence interval for the Pharmaceuticals, Reata Pharmaceuticals, Bayer Healthcare, Novartis Pharma, geometricmeanchange(%).Thesecondaryefficacy analysis of and AbbVie; all compensations are paid to his institution for research and change from baseline to final on-treatment log UACR was deter- educational activities. S.W.T. is a consultant for AbbVie and receives honoraria for academic talks from Servier and Valeant; he is an investigator on both mined by an analysis of covariance model with treatment and coun- contract and investigator-initiated research projects with AstraZeneca, Bristol- try (study 1 only) as the main effects and baseline measurement as Myers Squibb, Mitsubishi, and Pfizer. R.T. serves on advisory boards for AbbVie, the covariate. Similar statistical models were used to assess treat- Boehringer Ingelheim, Amgen, Eli Lilly, Takeda, Reata Pharmaceuticals, and fi ment group differences in other ef cacy and safety variables, such Merck & Co. G.V. is a consultant for GlaxoSmithKline, AbbVie, Mitsubishi as eGFR, serum creatinine, SBP, DBP, weight, hemoglobin, and Pharma, Daiichi Sankyo, Roche, and Eli Lilly. H.-H.P.is a consultant for AbbVie. lipids (cholesterol, LDL cholesterol, HDL cholesterol, and triglyc- B.C., D.A., J.J.B., H.T.,and M.H. are all AbbVie employees and own AbbVie stock. erides). Treatment group differences in the proportion of partici- pants that achieved at least a 30%, 40%, or 50% reduction from baseline to final on-treatment UACR was analyzed by the Fisher’s REFERENCES exact test. Concomitant medications including RAS inhibitors, di- uretics, b-blockers, calcium channel blockers, glucose-lowering 1. Heerspink HJ, de Zeeuw D: The kidney in type 2 diabetes therapy. Rev drugs, and lipid-modifying drugs were summarized by treatment Diabet Stud 8: 392–402, 2011 groups. 2. de Zeeuw D, Remuzzi G, Parving HH, Keane WF, Zhang Z, Shahinfar S, Snapinn S, Cooper ME, Mitch WE, Brenner BM: Proteinuria, a target for renoprotection in patients with type 2 diabetic nephropathy: Lessons from RENAAL. Kidney Int 65: 2309–2320, 2004 3. Atkins RC, Briganti EM, Lewis JB, Hunsicker LG, Braden G, Champion ACKNOWLEDGMENTS de Crespigny PJ, DeFerrari G, Drury P, Locatelli F, Wiegmann TB, Lewis EJ: Proteinuria reduction and progression to renal failure in patients with type 2 diabetes mellitus and overt nephropathy. Am J Kidney Dis Theauthorsgreatly appreciatethe participationofmany individuals in 45: 281–287, 2005 these studies as well as the efforts of all of the investigators. D.D.Z., 4. Cravedi P, Ruggenenti P, Remuzzi G: Proteinuria should be used as a R.C.-R., D.K., H.J.L.H., H.M., V.P.,G.R., S.W.T., R.T., G.V.,and H.-H.P. surrogate in CKD. Nat Rev Nephrol 8: 301–306, 2012 are members of the steering committee. Rajiv Agarwal, David Webb, 5. Ibsen H, Olsen MH, Wachtell K, Borch-Johnsen K, Lindholm LH, Rudolph Bilous, Mark Molitch, and Sheryl Kelsey are members of data Mogensen CE: Reduction in albuminuria translates to reduction in cardiovascular events in hypertensive patients with left ventricular hy- safety and monitoring board. pertrophy and diabetes. JNephrol21: 566–569, 2008 This study was supported by AbbVie. The sponsor was involved in 6. Schmieder RE, Mann JF, Schumacher H, Gao P, Mancia G, Weber MA, thedesignofthestudy,inthecollectionandanalysisofthedata,andin McQueen M, Koon T, Yusuf S; ONTARGET Investigators: Changes in writing the report. All authors had access to study results, and the lead albuminuria predict mortality and morbidity in patients with vascular JAmSocNephrol – author vouches for the accuracy and completeness of the data disease. 22: 1353 1364, 2011 fi 7. Kohan DE: Endothelin, hypertension and chronic kidney disease: New reported. The lead author had the nal decision to submit the insights. Curr Opin Nephrol Hypertens 19: 134–139, 2010 publication. The study was overseen by a data review committee. 8. Mann JF, Green D, Jamerson K, Ruilope LM, Kuranoff SJ, Littke T, Medical writing support was provided by Adebusola Ajibade, on Viberti G; ASCEND Study Group: Avosentan for overt diabetic ne- behalf of AbbVie. phropathy. JAmSocNephrol21: 527–535, 2010 An abstract containing data from this study was submitted and 9. Ko Kohan DE, Pollock DM: Endothelin antagonists for diabetic and non- Br J Clin Pharmacol – – diabetic chronic kidney disease. 76: 573 579, 2013 accepted for presentation at the European Renal Association European 10. Gagliardini E, Corna D, Zoja C, Sangalli F, Carrara F, Rossi M, Conti S, Dialysis and Transplant Association 2013 Congress, May 18-21, 2013, Rottoli D, Longaretti L, Remuzzi A, Remuzzi G, Benigni A: Unlike each Istanbul, Turkey. drug alone, lisinopril if combined with avosentan promotes regression

10 Journal of the American Society of Nephrology J Am Soc Nephrol 25: ccc–ccc,2014 www.jasn.org CLINICAL RESEARCH

of renal lesions in experimental diabetes. Am J Physiol Renal Physiol blockade reduces albuminuria in diabetic nephropathy. JAmSoc 297: F1448–F1456, 2009 Nephrol 22: 763–772, 2011 11. Simonson MS, Ismail-Beigi F: Endothelin-1 increases collagen accu- 17. AbbVie: A prospective, double-blind, placebo-controlled, multicenter mulation in renal mesangial cells by stimulating a chemokine and study to evaluate efficacy and safety of atrasentan, including thoracic cytokine autocrine signaling loop. J Biol Chem 286: 11003–11008, bioimpedance, in type 2 diabetic subjects with nephropathy. Available 2011 at: http://clinicaltrials.gov/ct2/show/NCT01399580?term=atrasentan+ 12. Wenzel RR, Littke T, Kuranoff S, Jürgens C, Bruck H, Ritz E, Philipp T, and+bioimpedance&rank=1. Accessed October 10, 2013 Mitchell A; SPP301 (Avosentan) Endothelin Antagonist Evaluation in 18. de Zeeuw D, Gansevoort RT, Dullaart RP, de Jong PE: Angiotensin II Diabetic Nephropathy Study Investigators: Avosentan reduces albu- antagonism improves the lipoprotein profile in patients with nephrotic min in diabetics with macroalbuminuria. J Am Soc Nephrol syndrome. J Hypertens Suppl 13: S53–S58, 1995 20: 655–664, 2009 19. Dhaun N, MacIntyre IM, Kerr D, Melville V, Johnston NR, Haughie S, 13. Galiè N, Hoeper MM, Gibbs JS, Simonneau G: Liver toxicity of sitaxentan Goddard J, Webb DJ: Selective endothelin-A receptor antagonism in pulmonary arterial hypertension. Eur Respir J 37: 475–476, 2011 reduces proteinuria, blood pressure, and arterial stiffness in chronic 14. Fattinger K, Funk C, Pantze M, Weber C, Reichen J, Stieger B, Meier PJ: proteinuric kidney disease. Hypertension 57: 772–779, 2011 The endothelin antagonist inhibits the canalicular bile salt 20. AbbVie: Randomized, multicountry, multicenter, double-blind, par- export pump: A potential mechanism for hepatic adverse reactions. allel, placebo-controlled study of the effects of atrasentan on renal Clin Pharmacol Ther 69: 223–231, 2001 outcomes in subjects with type 2 diabetes and nephropathy SONAR: 15. Opgenorth TJ, Adler AL, Calzadilla SV, Chiou WJ, Dayton BD, Dixon Study of Diabetic Nephropathy with Atrasentan. Available at: http:// DB, Gehrke LJ, Hernandez L, Magnuson SR, Marsh KC, Novosad EI, clinicaltrials.gov/ct2/show/NCT01858532?term=sonar&rank=1. Ac- Von Geldern TW, Wessale JL, Winn M, Wu-Wong JR: Pharmacological cessed October 10, 2013 characterization of A-127722: An orally active and highly potent ETA- selective receptor antagonist. J Pharmacol Exp Ther 276: 473–481, 1996 16. Kohan DE, Pritchett Y, Molitch M, Wen S, Garimella T, Audhya P, This article contains supplemental material online at http://jasn.asnjournals. Andress DL: Addition of atrasentan to renin-angiotensin system org/lookup/suppl/doi:10.1681/ASN.2013080830/-/DCSupplemental.

J Am Soc Nephrol 25: ccc–ccc, 2014 Atrasentan Reduces Macroalbuminuria 11 Supplementary Materials

The endothelin antagonist atrasentan lowers residual albuminuria in type 2 diabetic patients with nephropathy

Dick de Zeeuw et al.

Methods

Inclusion Criteria

A subject was selected for study participation if he or she met the following criteria:

1. Subject was at least 18 years of age. 2. Subject voluntarily signed and dated an informed consent form, approved by an IRB/IEC, after the nature of the study was explained and the subject had the opportunity to ask questions. The informed consent had to be signed before any study-specific procedures were performed.

3. Subject had type 2 diabetes and had been treated with at least 1 anti-hyperglycemic medication within the 12 months prior to the Screening Period.

4. Subject was currently receiving an ACEi or ARB (RAS inhibitor). 5. If female, subject was not breastfeeding and was not pregnant (verified by negative serum pregnancy test prior to the Treatment Period). Subject was not of childbearing potential, defined as postmenopausal for at least 1 year or surgically sterile (bilateral tubal ligation, bilateral oophorectomy, or hysterectomy) or was of childbearing potential and practicing 1 of the following methods of birth control: • Double-barrier method (any 2 of the following: condoms, contraceptive sponge, diaphragm with spermicidal jellies or creams). • Hormonal contraceptives (oral, parenteral, or transdermal) for at least 3 months prior to and during study drug administration or intrauterine device [IUD] and one barrier method (condoms, contraceptive sponge, and diaphragm with spermicidal jellies or creams). • Barrier method (condoms, contraceptive sponge, and diaphragm with spermicidal jellies or creams) with a vasectomized partner.

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• Total abstinence from sexual intercourse during the study (minimum one complete menstrual cycle prior to study start). Contraception had to be used during the study and for 4 weeks after the last dose of study drug.

6. For entry into the Run-In Period the subject had to satisfy the following criteria based on the screening laboratory values: • Estimated GFR ≥ 30 and ≤ 75 mL/min/1.73m2 by EPI formula • UACR ≥ 300 and ≤ 3,500 mg/g as determined by the geometric mean of the 2 morning void urine specimens obtained at the Screening visit (UACR ≥ 34 mg/mmol and ≤ 396 mg/mmol) • Serum albumin ≥ 3.0 g/dL (30 g/L) • BNP ≤ 200 pg/mL (57.8 pmol/L) • Negative serum pregnancy test for female subjects • SBP ≥ 110 mmHg and ≤ 180 mmHg

• HbA1c ≤ 12% 7. For entry into the Treatment Period the subject had to satisfy the following criteria based on the last visit of the Run-In Period laboratory values: • RAS inhibitor at maximum tolerated labeled dose for the previous 4 weeks with no adjustments of dose • Diuretic at any dose unless medically contraindicated (with the exception of loop diuretics ≥ 120 mg QD of furosemide or ≥ 3.0 mg QD of bumetanide or ≥ 150 mg QD of ethacrynic acid or ≥ 60 mg QD of torasemide) • UACR ≥ 200 mg/g as determined by the median of the 3 morning void urine specimens obtained prior to the Week –1 visit (UACR ≥ 23 mg/mmol) • SBP ≥ 110 mmHg and ≤ 160 mmHg • Serum potassium ≤ 5.5 mEq/L (5.5 mmol/L) • Negative serum pregnancy test for female subjects

The rationale for the inclusion criteria were as follows: the safety of the subjects (#1), compliance with harmonized GCP (#2), selection of an adequate subject population with appropriate disease severity for evaluation (#3, #4, #6, #7), and unknown impact of atrasentan on pregnancies (#5).

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Exclusion Criteria

A subject was to be excluded from the study if he/she met any of the following criteria:

1. Subject had a history of moderate or severe edema, facial edema unrelated to trauma, or a history of myxedema in the 6 months prior to Screening.

2. Subject was receiving loop diuretics ≥ 120 mg QD of furosemide or ≥ 3.0 mg QD of bumetanide or ≥ 150 mg QD of ethacrynic acid or ≥ 60 mg QD of torasemide.

3. Subject had a history of pulmonary edema. 4. Subject had a history of pulmonary hypertension or any lung diseases requiring (i.e., chronic obstructive pulmonary disease, emphysema, pulmonary fibrosis).

5. Subject had a documented history of heart failure, defined as American College of Cardiology/American Heart Association Practice Guidelines1:

• Stage C: structural heart disease with prior or current symptoms of heart failure or • Stage D: refractory heart failure requiring specialized interventions. 6. Subject had a history of orthostatic hypotension within the past 6 months as defined by the presence of a supine-to-standing blood pressure decrease ≥ 20 mmHg systolic or ≥ 10 mmHg diastolic within 3 minutes of standing.

7. Subject has a body mass index (BMI) > 45 at Screening Week –14. 8. Subject had an upper arm circumference > 42 cm (cuff for the ambulatory blood pressure measurement).

9. Subject had elevated liver enzymes (serum alanine aminotransaminase [ALT] and/or serum aspartate aminotransaminase [AST]) > 3 × the upper limit of normal (ULN) at Screening Week –14.

10. Subject had a hemoglobin < 9 g/dL at Screening Week –14. 11. Subject had a sensitivity to loop diuretics. 12. Subject had a history of an allergic reaction or significant sensitivity to atrasentan (or its excipients) or similar compounds.

13. Subject had a history of chronic gastrointestinal (GI) disease, which in the investigator's opinion may cause significant GI malabsorption. 3

14. Subject had a history of secondary hypertension (i.e., renal artery stenosis, primary aldosteronism or pheochromocytoma).

15. Subject had significant comorbidities (e.g., advanced malignancy, advanced liver disease) with a life expectancy less than 1 year.

16. Subject had clinically significant coronary artery disease (CAD) within 3 months prior to the Screening Period, defined as one of the following: • Hospitalization for myocardial infarction or unstable angina; or • New onset angina with positive functional study or coronary angiogram revealing stenosis; or • Coronary revascularization procedure. 17. Subject had a history of drug or alcohol abuse within 6 months prior to the Screening visit. 18. Subject had received any investigational drug within 3 months prior to Screening. 19. Subject received dialysis treatments or is expected to receive dialysis within 6 months. 20. Subject was currently receiving a combination of ACEi and ARB, rosiglitazone, aliskiren, or aldosterone blockers.

21. Subject was currently receiving pioglitazone and edema was present. The rationale for the exclusion criteria were as follows: the ensure the safety of the subjects (#1, #3-#19) and to avoid bias for the evaluation of efficacy and safety by the concomitant use of other medications (#2, #20, #21).

Assessment of Edema

Edema assessment was specified in the protocol as follows:

Assessment of edema was performed at every visit during the Screening, Run-In, Enrichment and Treatment Periods as well as the Follow-Up Visit. The assessment of edema was performed by the same site personnel at approximately the same time (if possible) at each visit and the time of assessment was recorded. The severity of edema for each subject was defined by the following categories:

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• None: edema is not present on examination; • Mild: edema is present on examination, but asymptomatic and the subject is willing to continue study medication; • Moderate: edema is present on examination, but symptomatic and the subject is willing to continue study medication; • Severe: edema is present on examination, but symptomatic and the subject is unwilling to continue study medication, or subject has an adverse event of pulmonary edema or congestive heart failure (CHF).

New onset or worsening edema, as deemed by the investigator, was captured as adverse events. The date of edema resolution was also captured.

Suppl. Figure 1. Placebo-corrected change in eGFR after 0.75 (■) and 1.25 mg/day (▲) atrasentan treatment and after recovery.

5

Suppl. Table 1. Demographics and baseline characteristics of the intent-to-treat population for RADAR study (M11-350, CT.gov NCT01356849). Data are presented as mean (SD) unless otherwise noted.

Atrasentan

Placebo 0.75 mg 1.25 mg

(n=30) (n=59) (n=64)

Age, years 63.0 (8.5) 64.3 (9.6) 64.0 (9.0)

Gender, n (%)

Male 22 (73) 49 (83) 47 (73)

Female 8 (27) 10 (17) 17 (27)

Race, n (%)

White 23 (77) 36 (61) 38 (59)

Black 2 (7) 14 (24) 13 (20)

Asian 4 (13) 6 (10) 9 (14)

Other 1 (3) 3 (5) 4 (6)

Ethnicity, n (%)

Hispanic or Latino 10 (33) 17 (29) 23 (36)

Other 20 (67) 42 (71) 41 (64)

Weight, kg 94.8 (18.1) 92.8 (21.5) 92.7 (17.3)

Known duration of diabetes, years 14.9 (8.2) 15.4 (9.8) 17.2 (10.5)

SBP, mm Hg 137 (15) 138 (14) 136 (14)

DBP, mm Hg 71 (10) 74 (10) 74 (9)

Serum albumin, g/l 40.8 (3.3) 40.3 (3.6) 40.3 (3.3)

Serum creatinine, mg/dl 1.60 (0.4) 1.70 (0.45) 1.50 (0.35)

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eGFR, ml/min/1.73 m2 46.4 (13.4) 46.0 (14.1) 48.7 (12.2)

Hemoglobin, g/l 12.3 (1.6) 12.7 (1.3) 12.8 (1.7)

HbA1c, % 7.9 (1.2) 7.8 (1.5) 8.0 (1.5)

Total cholesterol, mg/dl 173 (49) 161 (39) 164 (37)

LDL cholesterol, mg/dl 89 (39) 82 (32) 80 (27)

HDL cholesterol, mg/dl 46 (12) 44 (13) 44 (10)

Triglycerides, mg/dl 180 (94) 184 (140) 199 (122)

Serum potassium, mmol/l 4.63 (0.51) 4.51 (0.53) 4.49 (0.53)

UACR, median (Q1 to Q3), mg/g 633 853 817 creatinine (406 to 1295) (526 to 1682) (425 to 1398)

Antihypertensives, n (%)

RAS inhibitors 30 (100) 59 (100) 64 (100)

Beta blockers, n (%) 16 (53) 27 (46) 37 (58)

Calcium channel blockers, n (%) 14 (47) 27 (46) 29 (45)

Diuretics, n (%)

Loop diuretics 16 (53) 23 (39) 19 (30)

Thiazides 12 (40) 29 (49) 29 (45)

Glucose-lowering therapies, n (%)

Insulin glargine 11 (37) 23 (39) 22 (34)

Metformin 5 (17) 14 (24) 16 (25)

Sulphonylurea 14 (47) 25 (42) 24 (38)

Statins, n (%) 23 (77) 47 (80) 53 (83)

CADa, n (%) 7 (23) 13 (22) 2 (3)

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Stroke, n (%) 4 (13) 4 (7) 8 (9)

aCoronary Artery Disease (CAD)

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Suppl. Table 2. Demographics and baseline characteristics of the intent-to-treat population for JAPAN study (M12-812, CT.gov NCT01424319). Data are presented as mean (SD) unless otherwise noted.

Atrasentan

Placebo 0.75 mg 1.25 mg

(n=20) (n=19) (n=19)

Age, years 66.4 (9.5) 67.3 (10.3) 66.0 (8.1)

Gender, n (%)

Male 18 (90) 14 (74) 10 (53)

Female 2 (10) 5 (26) 9 (47)

Race, n (%)

White - - -

Black - - -

Asian 20 (100) 19 (100) 19 (100)

Other - - -

Ethnicity, n (%)

Hispanic or Latino - - -

Other 20 (100) 19 (100) 19 (100)

Weight, kg 68.6 (11.0) 69.3 (12.6) 73.4 (13.9)

Known duration of diabetes, years 14.0 (11.3) 14.9 (8.1) 15.9 (4.9)

SBP, mm Hg 132 (14) 138 (17) 133 (15)

DBP, mm Hg 72 (10) 75 (13) 76 (11)

Serum albumin, g/l 39.1 (5.1) 40.5 (4.0) 41.4 (3.0)

Serum creatinine, mg/dl 1.40 (0.31) 1.40 (0.35) 1.20 (0.27)

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eGFR, ml/min/1.73 m2 53.7 (12.1) 53.8 (15.1) 56.9 (16.3)

Hemoglobin, g/l 13.4 (2.0) 13.2 (1.8) 13.1 (2.2)

HbA1c, % 6.7 (1.0) 6.5 (1.1) 6.7 (0.6)

Total cholesterol, mg/dl 196 (44) 202 (37) 197 (33)

LDL cholesterol, mg/dl 115 (36) 113 (28) 111 (28)

HDL cholesterol, mg/dl 48 (11) 52 (15) 51 (17)

Triglycerides, mg/dl 144 (60) 177 (97) 173 (72)

Serum potassium, mmol/l 4.62 (0.48) 4.65 (0.54) 4.56 (0.43)

UACR, median (Q1 to Q3), mg/g 1454 960 961 creatinine (456 to 2474) (401 to 2113) (505 to 1356)

Antihypertensives, n (%)

RAS inhibitors 20 (100) 19 (100) 19 (100)

Beta blockers, n (%) - 4 (21) 1 (5)

Calcium channel blockers, n (%) 27 (54) 42 (54) 46 (55)

Diuretics, n (%)

Loop diuretics 3 (15) 6 (32) 8 (42)

Thiazides 17 (85) 13 (68) 14 (74)

Glucose-lowering therapies, n (%)

Insulin glargine 1 (5) 2 (11) 1 (5)

Metformin 8 (40) 5 (26) 6 (32)

Sulphonylurea 13 (65) 8 (42) 8 (42)

Statins, n (%) 15 (75) 11 (58) 15 (79)

CADa, n (%) 1 (5) - 1 (5)

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Stroke, n (%) 6 (30) 4 (21) 6 (32)

aCoronary Artery Disease (CAD)

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Suppl. Table 3. Summary of incidence of adverse events for RADAR study (M11-350, CT.gov NCT01356849). Placebo Atrasentan

0.75 mg 1.25 mg

(n=30) (n=59) (n=64)

Any Adverse Event 20 (67%) 38 (64%) 47 (73%)

Any Serious Adverse Event 3 (10%) 4 (7%) 4 (6%)

Peripheral Edema 15 (50%) 20 (34%) 27 (42%)

Facial Edema - - -

Congestive Heart Failure 1 (3.3%) 1 (1.7%) -

Weight Increased 1 (3.3%) - 3 (4.7%)

Headache 2 (6.7%) 1 (1.7%) 2 (3.1%)

Nasal congestion and Rhinitis 2 (6.7%) - 1 (1.6%)

Diarrhea and Constipation 5 (17%) 7 (12%) 7 (11%)

Deaths - - -

Adverse Event Leading to - 5 (9%) 7 (11%) Discontinuation of Study Drug

Peripheral Edema - 2 (3.4%) 3 (4.7%)

Facial Edema - - -

Anemia - - -

Fatigue - 1 (1.7%) 2 (3.1%)

Hyperkalemia - - 1 (1.6%)

Helicobacter pylori infection - 1 (1.7%) -

Hypoglycemia - - 1 (1.6%)

Coronary Artery Stenosis - - 1 (1.6%)

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Acute Appendicitis - 1 (1.7%) -

Lung Hemorrhage - - -

Thyroid Cancer - - -

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Suppl. Table 4. Summary of incidence of adverse events for JAPAN study (M12-812, CT.gov NCT01424319). Placebo Atrasentan

0.75 mg 1.25 mg

(n=20) (n=19) (n=19)

Any Adverse Event 14 (70%) 17 (90%) 14 (74%)

Any Serious Adverse Event 1 (5%) 3 (16%) 2 (11%)

Peripheral Edema 6 (30%) 7 (37%) 8 (42%)

Facial Edema - - 3 (16%)

Congestive Heart Failure - - -

Weight Increased - - -

Headache - - 3 (16%)

Nasal congestion and Rhinitis - - -

Diarrhea and Constipation - - 2 (11%)

Deaths - - -

Adverse Event Leading to - 1 (5%) 5 (26%) Discontinuation of Study Drug

Peripheral Edema - - 1 (5%)

Facial Edema - - 2 (11%)

Anemia - - 2 (11%)

Fatigue - - -

Hyperkalemia - - -

Helicobacter pylori infection - - -

Hypoglycemia - - -

Coronary Artery Stenosis - - -

14

Acute Appendicitis - - -

Lung Hemorrhage - 1 (%) -

Thyroid Cancer - - 1 (%)

References

1. Hunt, SA, Abraham, WT, Chin, MH, Feldman, AM, Francis, GS, Ganiats, TG, Jessup, M, Konstam, MA, Mancini, DM, Michl, K, Oates, JA, Rahko, PS, Silver, MA, Stevenson, LW, Yancy, CW: 2009 focused update incorporated into the ACC/AHA 2005 Guidelines for the Diagnosis and Management of Heart Failure in Adults: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines: developed in collaboration with the International Society for Heart and Lung Transplantation. Circulation, 119: e391-479, 2009.

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