Addition of Atrasentan to Renin-Angiotensin System Blockade Reduces Albuminuria in Diabetic Nephropathy

CLINICAL RESEARCH www.jasn.org

Donald E. Kohan,* Yili Pritchett,† Mark Molitch,‡ Shihua Wen,† Tushar Garimella,† Paul Audhya,† and Dennis L. Andress†

*Division of Nephrology, Department of Medicine, University of Utah Health Sciences Center, Salt Lake City, UT; †Abbott Laboratories, Abbott Park, IL; and ‡Division of Endocrinology, Metabolism and Molecular Medicine, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL

ABSTRACT Although endothelin-receptor antagonists reduce albuminuria in diabetic nephropathy, fluid retention limits their use. Here, we examined the effect of atrasentan, a selective endothelin A receptor (ETAR) antagonist, on albuminuria in a randomized, double-blind, placebo-controlled trial of subjects with diabetic nephropathy already receiving stable doses of renin-angiotensin system (RAS) inhibitors. We randomly assigned 89 subjects with eGFR Ͼ20 ml/min per 1.73 m2 and a urinary albumin-to-creatinine ratio (UACR) of 100 to 3000 mg/g to placebo or atrasentan (0.25, 0.75, or 1.75 mg daily) for 8 weeks. Compared with placebo, atrasentan significantly reduced UACR only in the 0.75- and 1.75-mg groups (P ϭ 0.001 and P ϭ 0.011, respectively). Compared with the 11% reduction in the geometric mean of the UACR from baseline to final observation in the placebo group during the study, the geometric mean of UACR decreased by 21, 42, and 35% in the 0.25-, 0.75-, and 1.75-mg atrasentan groups (P ϭ 0.291, P ϭ 0.023, and P ϭ 0.073, respectively). In the placebo group, 17% of subjects achieved Ն40% reduction in UACR from baseline compared with 30, 50, and 38% in the 0.25-, 0.75-, and 1.75-mg atrasentan groups, respectively (P ϭ 0.029 for 0.75 mg versus placebo). Peripheral edema occurred in 9% of subjects receiving placebo and in 14, 18, and 46% of those receiving 0.25, 0.5, and 1.75 mg atrasentan, respectively (P ϭ 0.007 for 1.75 mg versus placebo). In summary, atrasentan, at the doses tested, is generally safe and effective in reducing residual albuminuria and may ultimately improve renal outcomes in patients with type 2 diabetic nephropathy.

J Am Soc Nephrol 22: 763–772, 2011. doi: 10.1681/ASN.2010080869

Diabetic nephropathy (DN) continues to be the The endothelin (ET) system is chronically ac- most common cause of ESRD, despite attempts at tivated in patients with diabetes and in preclini- rigorous control of hyperglycemia and hyperten- cal models as evidenced by elevated circulating sion.1,2 The addition of renin-angiotensin system levels of endothelin-1 (ET-1),6 enhanced kidney

(RAS) inhibitors to reduce the deleterious effects ET-1 concentrations,7 and increased renal and CLINICAL RESEARCH of excessive renal angiotensin receptor activation systemic endothelin A receptor (ETAR) activa- has been the only kidney-specific therapy developed for DN during the past 10 years. Although Received August 23, 2010. Accepted November 22, 2010. treatment with RAS inhibitors shows reductions in albuminuria in association with delays in Published online ahead of print. Publication date available at www.jasn.org. chronic kidney disease (CKD) progression,3,4 Correspondence: Dr. Donald Kohan, Division of Nephrology, there remains a significant unmet need to de- University of Utah Health Sciences Center, 1900 East 30 North, velop therapies that completely prevent progres- Salt Lake City, UT 84132. Phone: 801-581-2726; Fax: 801-581- sion to ESRD or even induce regression of glo- 4343; E-mail: [email protected] merular pathology.5 Copyright © 2011 by the American Society of Nephrology

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8 tion. Glomerular ETAR, but not ETBR, activation promotes Patient Characteristics podocyte and mesangial cell dysfunction, leading to pro- Baseline demographic, clinical and biochemical characteris- teinuria and glomerulosclerosis.9 A recent clinical trial with tics, and concomitant therapies were balanced between the avosentan, an endothelin receptor antagonist that likely four groups (Table 1). At baseline, 27% of subjects had 30 to Ͼ blocked both ETAR and ETBR, reduced albuminuria in pa- 200 mg/g creatinine, 72% of subjects had 200 mg/g creati- tients with macroalbuminuria and type 2 diabetes, although nine, and 26% of subjects had an estimated GFR Ͼ60 ml/min significant safety concerns related to fluid retention resulted per 1.73 m2. The majority of subjects (87%) were white, and in early trial termination.10 the mean age of the study population was 64 years.

Atrasentan is a highly selective ETAR antagonist with an 11 approximate 1800:1 selectivity for ETARtoETBR. Such Primary and Secondary Outcomes ETAR, as opposed to ETBR, selectivity may be ideal for tar- The primary efficacy analysis, comparing treatment group dif- geting the ET pathogenicity in DN. The purpose of this ferences between each atrasentan group and placebo for randomized, double-blind, placebo-controlled clinical trial change from baseline to each postbaseline assessment (after a was to prospectively evaluate the efficacy and safety of atra- log transformation) using a repeated-measures analysis sentan for the reduction of residual albuminuria in subjects showed that urinary albumin-to-creatinine ratio (UACR) was with type 2 DN who were receiving stable doses of angioten- significantly reduced during the course of the 8-week treat- sin converting enzyme inhibitors (ACEIs) or angiotensin ment period in the 0.75- and 1.75-mg groups (P ϭ 0.001 and receptor blockers (ARBs). P ϭ 0.011 versus placebo, respectively; Figure 2). For the 0.75-mg group, a significant treatment effect was seen as early as week 1 (P ϭ 0.005) and was sustained to the last treatment RESULTS visit (week 8) of the study (P ϭ 0.008). The modest UACR reduction in the 0.25-mg group was not significant (P ϭ The disposition of study subjects is shown in Figure 1. Of the 0.150). 239 subjects screened, 89 subjects comprised the intent-to- Multiplicity adjustments were not made among the three treat population and were randomly assigned to one of four pairwise comparisons for the primary efficacy analysis because treatment groups: placebo (n ϭ 23), atrasentan 0.25 mg daily this was an exploratory phase 2a study. However, if a Bonfer- (n ϭ 22), 0.75 mg daily (n ϭ 22), or 1.75 mg daily (n ϭ 22). roni adjustment is made post hoc to adjust for multiplicity of

Figure 1. Disposition of subjects during the study. Subjects may have had more than one reason for discontinuation.

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Table 1. Subject demographics and baseline characteristics Atrasentan Placebo Variable mg 0.75 mg 1.75 mg 0.25 (23 ؍ n) (22 ؍ n) (22 ؍ n) (22 ؍ n) Gender, n (%) female 4 (17%) 9 (41%) 8 (36%) 6 (27%) male 19 (83%) 13 (59%) 14 (64%) 16 (73%) Race, n (%) white 22 (96%) 19 (86%) 18 (82%) 18 (82%) black 0 3 (14%) 2 (9%) 2 (9%) other 1 (4%) 0 2 (9%) 2 (9%) Ethnicity, n (%) Hispanic or Latino 13 (57%) 14 (64%) 14 (64%) 11 (50%) no ethnicity 10 (44%) 8 (36%) 8 (36%) 11 (50%) Age, years, n (%) Ͻ65 12 (52%) 13 (59%) 7 (32%) 11 (50%) Ն65 11 (48%) 9 (41%) 15 (68%) 11 (50%) Age, years mean (SD) 61 (8) 63 (12) 67 (9) 64 (13) Weight, kg mean (SD) 99 (20) 84 (13) 96 (19) 97 (20) Body mass index, kg/m2 mean (SD) 34 (5) 31 (4) 34 (6) 33 (5) UACR, mg/g creatinine median (Q1 to Q3) 515 (170 to 1477) 350 (194 to 1226) 360 (209 to 726) 433 (157 to 998) Estimated GFR, ml/min/BSA mean (SD) 52 (25) 50 (24) 61 (25) 48 (20) Serum creatinine, mg/dl Mean (SD) 1.6 (0.6) 1.5 (0.6) 1.3 (0.5) 1.8 (0.8) SBP, mmHg mean (SD) 138 (14) 134 (14) 137 (15) 135 (11) DBP, mmHg mean (SD) 78 (8) 75 (8) 74 (8) 75 (9) Hemoglobin, g/dl mean (SD) 13 (1) 12 (1) 13 (2) 13 (1) Hemoglobin A1c, % mean (SD) 7.4 (0.9) 7.6 (1.0) 7.6 (1.2) 7.3 (1.1)

comparisons among three pairs, the study can still claim suc- ment groups: 0.25 mg, 40% (P ϭ 0.999); 0.75 mg, 68% (P ϭ cess because the significance level of 0.017 (0.05/3) was 0.075); 1.75 mg, 62% (P ϭ 0.227). achieved by the prespecified primary efficacy analysis in the There was an early and sustained reduction in systolic BP 0.75 mg (P ϭ 0.001 versus placebo) and 1.75 mg groups (P ϭ (SBP) in the 0.75-mg group (P ϭ 0.038 by repeated-measures 0.011 versus placebo). analysis versus placebo) as shown in Figure 4A. The mean The geometric mean reduction from baseline to final UACR change from baseline to week 8 of treatment SBP was Ϫ0.3 was significantly greater in the 0.75-mg group (42% reduction) mmHg in the 0.25-mg group (P ϭ 0.834 versus 0.7 mmHg in compared with placebo (11% reduction, P ϭ 0.023). For the placebo), Ϫ8.8 mmHg (P ϭ 0.049 versus placebo) in the 1.75-mg group, the effect did not quite meet significance (35% 0.75-mg group, and Ϫ7.6 mmHg (P ϭ 0.086 versus placebo) in reduction, P ϭ 0.073), whereas the reduction by the 0.25-mg the 1.75-mg group. There was also an early and sustained de- group (21% reduction, P ϭ 0.291) was not significant com- crease in diastolic BP (DBP; Figure 4B) in the same treatment pared with placebo. A significantly greater proportion of sub- groups (Figure 4B), where the mean change from baseline to jects in the 0.75-mg group achieved at least a 40% reduction week 8 of treatment was Ϫ0.5 mmHg in the 0.25-mg group from baseline to final UACR compared with placebo (50 versus (P ϭ 0.753 versus Ϫ1.4 mmHg in placebo) Ϫ5.8 mmHg in the 17%, P ϭ 0.029; Figure 3). The proportion achieving Ն40% 0.75-mg group (P ϭ 0.132 versus placebo), and Ϫ7.4 mmHg in reduction in UACR in the 1.75- and 0.25-mg groups was 38 the 1.75-mg group (P ϭ 0.042 versus placebo). (P ϭ 0.179) and 30% (P ϭ 0.473), respectively. The proportion Linear regression models (path analysis) to study the rela- of subjects achieving Ն25% reduction was not significantly tionship between change in SBP and change in log UACR sug- different compared with placebo (39%) for any of the treat- gested that the SBP response was associated with only a minor

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Figure 2. Atrasentan treatment significantly reduces albuminuria. Effect of atrasentan on change in UACR from baseline. Significant reductions in UACR were seen with the 0.75-mg dose (P ϭ 0.001 versus placebo by repeated measures analysis) and 1.75-mg dose (P ϭ 0.011 versus placebo by repeated-measures analysis). UACR returned toward baseline values after 30 days from drug discontinuation.

Figure 4. Atrasentan affects longitudinal measures of BP by repeated-measures analysis. Systolic BP was reduced in the 0.75-mg dose (P ϭ 0.038 versus placebo by repeated-measures analysis). Diastolic BP was reduced in the 0.75-mg dose (P ϭ 0.017 versus placebo by repeated-measures analysis). BP values returned toward baseline after 30 days from drug discontinuation.

Figure 3. Atrasentan treatment significantly increases the per- All subjects received concomitant RAS inhibitors per the centage of subjects achieving Ն 40% reduction in UACR compared to placebo. study inclusion criteria. Thirty-eight percent of subjects received the maximum dose as recommended by the product portion of the UACR reduction (Ͻ21% of the total treatment label. To evaluate whether concomitantly taking the maximum effect). There were no significant differences in the mean dose of RAS inhibitors could influence the effect of atrasentan change from baseline in estimated GFR (range, Ϫ2 to 2 ml/min on UACR, a post hoc analysis was conducted. Subjects were per 1.73 m2 for all three dose groups across postbaseline visits) dichotomized by those who received maximum doses of RAS or body weight (range, Ϫ1.0 to 1.1 kg for all three dose groups inhibitors versus those who did not; the treatment-by-sub- across postbaseline visits; Figure 1; Appendix) between atra- group interaction on log UACR for change from baseline to sentan dose groups and placebo. There were significant reduc- final observation was not significant (P ϭ 0.816), indicating tions in hemoglobin concentrations induced by atrasentan that the treatment effect of atrasentan was present regardless of (Figure 2; Appendix), consistent with the known vasodilatory the level of RAS inhibition (Figure 3; Appendix). In the same and thus hemodilutional effect of this class of compounds. The model for subgroup analysis, the effect for subgroup was not mean change from baseline to week 8 of treatment was Ϫ0.7 statistically significant. g/dl in the 0.25-mg group, Ϫ0.4 g/dl in the 0.75-mg group, and Pharmacokinetic parameters for atrasentan on day 1 of Ϫ0.9 g/dl in the 1.75-mg group compared with 0.1 g/dl for treatment and the mean concentrations at each of the visits placebo (P Ͻ 0.001, P ϭ 0.015, and P Ͻ 0.001, respectively). (weeks 2, 4, 6, and 8) are presented in Tables 2 and 3, respec- All of the changes in UACR, BP, and hemoglobin returned tively. toward baseline values at the 30-day follow-up visit in the atra- Diuretic use (48 to 68%) was similar among the treat- sentan 0.75-mg and 1.75-mg groups. ment groups throughout the study (11, 12, 15, and 11 sub-

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Table 2. Atrasentan pharmacokinetic parameters on treatment day 1 Adverse Events Atrasentan There was a significantly higher proportion Pharmacokinetic of adverse events considered to be at least Parameters (units) 0.25 mg 0.75 mg 1.75 mg possibly related to study drug only in the (21 ؍ n) (23 ؍ n) (20 ؍ n) Ϯ Ϯ Ϯ 1.75-mg group (59 versus 22% in placebo, Tmax (h) 2.19 2.66 1.38 1.98 1.12 1.74 Ϯ Ϯ Ϯ P ϭ 0.016). Two subjects each from the 0.75- Cmax (ng/ml) 0.28 0.24 1.72 1.34 4.12 3.15 ⅐ Ϯ Ϯ Ϯ and 1.75-mg groups discontinued the drug AUC0–6 (ng h/ml) 0.71 0.91 5.31 3.83 13.07 9.74 Ϯ prematurely because of one or more of the Data presented as mean SD. Tmax, time to Cmax;Cmax, maximum plasma concentration; AUC0–6, area under the plasma concentration time curve from 0 to time to last sample. following adverse events: congestive heart failure and coronary artery disease (non- Table 3. Atrasentan trough plasma concentrations on treatment weeks 2, 4, emergent cardiac catheterization) in the 6, and 8 0.75-mg group and hypotension, angio- Treatment Visits Dose (mg) edema, headache, and peripheral edema in Week 2 Week 4 Week 6 Week 8 the 1.75-mg group. The most common ad- 0.25 0.52 Ϯ 0.28 0.63 Ϯ 0.47 0.54 Ϯ 0.48 0.46 Ϯ 0.26 verse event was peripheral edema, which 0.75 1.81 Ϯ 1.83 2.06 Ϯ 2.06 1.62 Ϯ 1.27 1.61 Ϯ 1.80 showed a dose–response relationship: pla- 1.75 2.22 Ϯ 1.05 2.28 Ϯ 1.10 2.25 Ϯ 1.06 2.39 Ϯ 1.14 cebo, 9%; 0.25 mg, 14%; 0.75 mg, 18%; Data presented as mean Ϯ SD. 1.75 mg, 46% (P ϭ 0.007 for placebo versus 1.75 mg). Peripheral edema was reported as Table 4. Treatment-emergent adverse events in study subjects mild in two subjects and moderate in two Atrasentan Placebo subjects in the 0.75-mg group and as mild Subjects Experiencing, N (%) -mg 0.75 mg 1.75 mg in nine subjects and moderate in one sub 0.25 (23 ؍ n) ject in the 1.75-mg group. One subject was (22 ؍ n) (22 ؍ n) (22 ؍ n) Any adverse event 13 (57%) 16 (73%) 16 (73%) 19 (86%)a discontinued from the study because of an- Any adverse event at least possibly 5 (22%) 8 (36%) 6 (27%) 13 (59%)b gioedema (in the 1.75-mg group). Com- related to study drug bining these two MedDRA preferred terms Any severe adverse event 0 1 (5%) 0 1 (5%) (peripheral edema and edema), the impact Any serious adverse event 0 1 (5%) 3 (14%) 1 (5%) of edema on subjects’ percentage change in Any adverse event leading to 0 0 2 (9%) 2 (9%) UACR was examined (Figure 5). The anal- discontinuation of study drug Deaths 0 0 0 0 ysis indicated that the treatment effect of Most commonly reported adverse atrasentan 0.75 mg was independent of the effectsc occurrence of edema. peripheral edema 2 (9%) 3 (14%) 4 (18%) 10 (46%)d Serious adverse events were observed in diarrhea 2 (9%) 1 (5%) 3 (14%) 0 0% of placebo, in 5% of the 0.25-mg group, dizziness 0 3 (14%) 2 (9%) 1 (5%) in 14% of the 0.75-mg group, and in 5% of urinary tract infection 1 (4%) 0 2 (9%) 3 (14%) the 1.75-mg group (Table 4). Only one seri- headache 0 2 (9%) 1 (5%) 2 (9%) ous adverse event was considered to be possi- cough 1 (4%) 1 (5%) 2 (9%) 0 bly related to study drug, occurring in a sub- hypertension 1 (4%) 1 (5%) 1 (5%) 1 (5%) ject in the 0.75-mg group who developed hypoglycemia 0 3 (14%) 0 1 (5%) accelerated hypertension and diastolic heart hypotension 1 (4%) 0 1 (5%) 2 (9%) failure approximately 3 weeks after starting aP ϭ 0.047 versus placebo. bP ϭ 0.016 versus placebo. treatment that was quickly reversed with BP cReported in Ն5% of subjects. control and diuretic therapy. This subject had d ϭ P 0.007 versus placebo. a baseline N-terminal pro-brain type natriuretic peptide (NT-pro BNP) NT-pro BNP jects in the placebo and 0.25-, 0.75-, and 1.75-mg groups, level that was Ͼ20-fold higher than normal before receiving atra- respectively). Loop diuretics (furosemide or torsemide) sentan, which may have reflected subclinical diastolic heart fail- were the most common type in 8, 5, 10, and 9 subjects, ure. The protocol was amended shortly thereafter to exclude sub- respectively (80 to 97% of treatment days), followed by dis- jects with a NT-pro BNP of 500 pg/ml or greater. tal diuretics (hydrochlorothiazide, chlorthalidone, or meto- There were two additional nonserious adverse cardiovascu- lazone) in 5, 9, 6, and 2 subjects, respectively (85 to 100% of lar events (atrioventricular block, coronary artery disease), one treatment days). Spironolactone was used in one placebo each in the 0.75-mg and 1.75-mg groups, which were consid- subject (all treatment days), and triamterene was used in ered by the investigator to not be related to the study drug. three subjects (one in 0.25 mg for 76 days and two in There were two nonserious adverse events of acute renal fail- 0.75-mg group for all treatment days). ure, both occurring in the 0.75-mg group. One occurred in a

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An acceptable safety profile of atrasentan treatment was observed in this trial, although the treatment period was relatively short. Only one serious adverse event related to fluid retention occurred in a subject with a history of cardiovascular disease. It is notable that this subject began treatment with a markedly elevated NT-pro- BNP that predicted an impending acute event,12 and it is therefore unclear what role atrasentan had in this case. Nevertheless, we believe that patients who are at high risk for developing congestive heart failure should be appropriately assessed for that

risk before initiating ETAR antagonist ther- Figure 5. The effect of atrasentan on change in UACR from baseline to final visit is apy. Although it was expected that periph- independent of edema occurrence during treatment. eral edema would constitute the majority of adverse events, it is reassuring that a clear subject with a baseline serum creatinine of 2.0 mg/dl, rising to dose–response relationship was seen, a low relative rate of edema 3.3 mg/dl, 5 days after an increase in diuretic dose and 7 days occurred with the atrasentan dose that had the greatest effect after starting treatment (corresponding to a BP change from on albuminuria reduction (0.75 mg), and most episodes were 127/73 to 115/60). Serum creatinine fluctuated between 3.3. mild in severity. Although mild edema was most common, and 2.4 through week 7 and then rose to 4.0 mg/dl at week 8 even with the highest dose studied, the frequency was signifi- (BP 113/80). The 30-day post-treatment creatinine and BP cantly higher than observed with placebo, and therefore 1.75 were 1.9 mg/dl and 145/64, respectively. The other event was mg of atrasentan may ultimately be a limiting therapeutic dose prerenal azotemia occurring after the subject, who had a his- because greater efficacy was not established compared with the tory of percutaneous transluminal coronary angioplasty, was 0.75-mg group. We attribute the low incidence of edema to the discontinued at study week 5 for nonemergent coronary artery doses studied and to atrasentan’s chemical structure, which

bypass grafting. provides an approximate 1800 fold selectivity for ETAR:ETBR. There were no significant changes in serum sodium, hepatic However, concerns about fluid retention will continue to be an enzymes, or bilirubin in any of the atrasentan treatment important management issue in patients taking ETR antago- groups compared with placebo. nists, and a better understanding of how both receptor systems are involved with sodium excretion continues to be elucidated.

Current evidence supports using a highly selective ETARan- DISCUSSION tagonist because of the importance of ETBR in mediating sodium excretion. The ETBR, located in the collecting duct, is 13 This study showed that atrasentan, a selective ETAR antagonist, crucial for normal sodium excretion. Studies in mice with is safe and efficacious in treating residual albuminuria over an collecting duct-specific knockout of ETAR and/or ETBR have 8-week period in subjects with type 2 DN who are on stable established that ETB receptors are of primary importance in doses of RAS inhibitors. The UACR-lowering effect, which oc- mediating ET-1 inhibition of renal sodium reabsorption13–16; curred early after drug initiation, was sustained throughout the inhibition of ETBR is highly likely, therefore, to cause fluid treatment period for the 0.75- and 1.75-mg doses but was not retention. ETAR blockade is also associated with vasodilatation significant in the lowest dose group. Although both of the ef- and may also promote fluid retention; however, the mecha- fective doses were associated with significant lowering of BP, nisms responsible for this, if it does occur, are poorly under- the major effect of atrasentan on UACR reduction was inde- stood. pendent of the BP change. The finding that estimated GFR did The clinical relevance of fluid retention with the use of not change during treatment suggests that ETAR antagonism ETAR antagonists in patients with diabetes and CKD became of efferent vasoconstriction may not be the dominant mecha- apparent in recent studies that examined the effect of avosen- nism for its albuminuria-lowering effect, in contrast to the tan on albuminuria lowering17 and on CKD progression.10 action of compounds that reduce renal angiotensin receptor In the dose ranging, 12-week study of albuminuria reduction, activation and decrease estimated GFR over a similar time treatment-emergent edema occurred in all of the avosentan frame.3,4 However, more definitive testing of changes in true treatment groups (5, 10, 25, and 50 mg) in a dose-related man- GFR and renal plasma flow will be necessary to determine ner (9 to 24% incidence) that was significantly greater than in whether filtration fraction is favorably altered in response to the placebo group (4%).17 In the larger hard endpoint trial, 45 atrasentan. and 46% of subjects in the avosentan 25- and 50-mg groups,

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respectively, reported symptomatic edema compared with safety profile observed in long-term studies with patients 31% in the placebo group (P Ͻ 0.0001) after a median treat- taking larger doses of atrasentan.23 ment period of 4 months. These high rates of edema were as- The results of this study are restricted to the population of sociated with a higher incidence of chronic heart failure–re- patients with type 2 diabetes who have residual albuminuria lated adverse events in the avosentan 25- (6%) and 50-mg while receiving RAS inhibitors and who have not had a history (4%) groups compared with placebo (2%), which was a major of heart failure. Several large clinical trials with drugs that in- reason for the study’s premature termination.10 Although the tervene in the RAS have shown that reductions in albuminuria mechanism of the edema formation is not fully understood, are associated with long-term renoprotective effects, and the one likely possibility is that the relatively high doses of this observed protection from ESRD may be independent of 3,4 ETAR antagonist may have induced partial blockade of renal changes in BP control. As a result, reductions in UACR have 9 ETBR despite its 50:1 selectivity of ETARtoETBR binding. In been proposed as a surrogate outcomes marker, and therefore, contrast, the doses of atrasentan in this study may have limited it is expected that the large magnitude of reductions in UACR the incidence of edema through incomplete ETAR blockade, observed in our study may translate into improved renal and allowing the endogenous ET-1 to bind at both ETAR and ETBR potentially cardiovascular outcomes in hard endpoint trials, sites, limiting the systemic vasodilatory effects expected with because a reduction in albuminuria by 30% of baseline within

unopposed ET-1 binding at ETBRs. the first 6 to 12 months of treatment in patients with kidney The finding that SBP and DBP were reduced was not unex- disease has been shown to predict long-term renal24 and car- 25 pected because it is known that ETAR-mediated vasoconstric- diovascular outcomes. These results will need to be con- tion may contribute to hypertension in CKD.18 However, the firmed in larger size, placebo-controlled, randomized clinical small sample size may have contributed to a larger mean trials. change in SBP observed in this study. Moreover, this study was In conclusion, atrasentan may offer an additional therapeu- not designed to look for a BP effect using more rigorous mon- tic benefit to the current standard of care using RAS inhibition itoring conditions, such as ambulatory BP measurements. Fu- for albuminuria reduction in patients with type 2 DN. Out- ture trials that use a run-in period to establish adequate BP come-driven clinical trials will ultimately be required to estab- control before baseline and include ambulatory BP monitor- lish its long-term safety and efficacy in slowing or preventing ing will be critical for observing the true effect of atrasentan on the progression to ESRD. change in BP.

ETAR antagonists likely act to reduce glomerular hypertension by decreasing efferent arteriolar vasoconstriction to re- CONCISE METHODS 19 duce filtration fraction. ETAR antagonists may directly atten- uate podocyte dysfunction through downregulation of TGF␤ Study Design and inhibition of macrophage infiltration.20 In a recent study This was a randomized, double-blind, placebo-controlled clinical 21 of a type 1 DN model, the addition of an ETAR antagonist to trial that enrolled subjects from 21 sites in the United States and RAS blockade in animals with established DN resulted in re- Puerto Rico between June 2009 and June 2010. There were 239 sub- duced albuminuria and regression of glomerulosclerosis. jects screened, and 89 subjects were randomly assigned to receive These changes coincided with increased podocyte nephrin ex- atrasentan or matching placebo for 8 weeks. Subjects were examined pression, decreased accumulation of TGF␤ and collagen III, at baseline, every week during the treatment phase, and 30 days after reduced macrophage infiltration, and increased expression of treatment withdrawal. BP and pulse (measured twice at each visit, and matrix metalloproteinase-9, the enzyme principally involved the average was used for the analyses), adverse events, concomitant with matrix degradation.21 medications and adherence to medication regimens, and blood Despite the controlled and double-blinded design of this chemistry were assessed at each visit. For each subject, two first morn- study, the limitations of small sample size, short duration ing void urine specimens collected on 2 consecutive days before each of treatment, and absence of ambulatory BP readings re- scheduled visit were collected, and the geometric mean of two UACR quire that a larger and longer study be performed for con- measures was calculated and used as the UACR value for the visit to be firmation. The study was also limited by the absence of included in data analysis. quantitative measures of renal hemodynamics to quantify Randomization was stratified into two strata defined by baseline potential changes in filtration fraction and renal vascular UACR (thresholds of 1000 and Ͼ1000 mg/g). The randomization resistance. The short duration of observation also precludes schedule was computer generated by the study sponsor, and the ran- identifying additional potential long-term safety concerns, domization was implemented using an Interactive Voice Response such as liver enzyme elevation, which have been identified System. Subjects were assigned in a 1:1:1:1 ratio to placebo and atra- with this class of compounds.22 We expect, however, that sentan doses of 0.25, 0.75, or 1.75 mg daily within each stratum. Study hepatic abnormalities will not be a concern because atrasen- medication was masked and packaged by the study sponsor and dis- tan’s chemical structure is not expected to induce hepatic tributed to sites through the Interactive Voice Response System as enzymes given that it is a carboxylic acid derivative rather masked study drug kits. The study investigator, study subjects, and than a sulfonamide derivative, and there is a shown liver study sponsor’s personnel involved with analysis and collection of

J Am Soc Nephrol 22: 763–772, 2011 Atrasentan Reduces Albuminuria 769 CLINICAL RESEARCH www.jasn.org study data were completely blinded to the subject’s treatment group der the plasma concentration time curve from 0 to time to last sample assignment during the study. Subjects randomization assignments were calculated for concentration data on treatment day 1 using non- were not disclosed until all database issues had been resolved and the compartmental methods. In, addition atrasentan plasma trough con- study database was locked. centrations were summarized for pharmacokinetic data on treatment weeks 2, 4, 6, and 8. Subjects Eligible subjects were enrolled if they had type 2 diabetes and had been Safety Evaluation taking anti-diabetic medications for at least 1 year before screening Safety endpoints were evaluated weekly and 30 days after treatment and had received a stable dose of an ACE inhibitor or ARB for at least withdrawal and included vital signs (e.g., SBP and DBP), body weight, Ͼ 2 2 months, had an estimated GFR 20 ml/min per 1.73 m by the liver enzymes, hemoglobin, and adverse events. Automated BP mea- abbreviated Modified Diet in Renal Disease formula, and a UACR surements were performed twice with the appropriate cuff size in the 26 between 100 and 3000 mg/g. Female subjects were postmenopausal nondominant arm at 2-minute intervals after 5 minutes of rest while for at least 1 year or were surgically sterile. The main exclusion criteria in the sitting position, and the mean value was recorded. included a history of significant peripheral edema, heart failure, pulmonary edema, or loop diuretic therapy of Ͼ60 mg/d of furosemide Statistical Analysis (or equivalent) and recent coronary arterial disease. The full list of The sample size of 20 per arm was planned to allow the study to have inclusion and exclusion criteria is found in Appendix 1. at least 85% power to detect a group difference of Ϫ0.42 in mean Subjects received their usual care for diabetes and cardiovascular change from baseline to final observation in log-transformed UACR protection. If the subject’s BP exceeded 130/80 mmHg, anti-hyper- between an atrasentan group and the placebo group (this difference tensive medication (not including RAS inhibitors) was increased or corresponds to a 34% between-group reduction of geometric mean added to obtain acceptable BP control based on current guidelines. change in UACR from baseline to final) at the one-sided significance Dose alterations of ACE inhibitors or ARBs were not allowed after level of 0.05. A common SD of 0.46 was assumed. The sample size was randomization. Diuretics were added for new onset or worsening adjusted to allow for one to two subjects per group without any UACR edema at the discretion of the investigator according to the protocol- data after randomization. specified guidelines; subjects not taking a loop diuretic could receive a All analyses were performed on the intent-to-treat population, loop diuretic; subjects taking a loop diuretic could receive increased which was comprised of the data from all randomized subjects who doses (50% increase suggested as the initial increment). The study had received at least one dose of study drug. The primary efficacy protocol was approved by an independent ethics committee and local analysis was a mixed-effect, maximum likelihood, repeated-measures and central review boards, and all subjects provided written informed analysis for change from baseline to each postbaseline assessment of consent. log UACR. The model contained the terms of treatment, visit, treatment-by-visit interaction, baseline measurements, and baseline-by- Efficacy Parameters visit interaction with unstructured as the variance-covariance struc- The protocol-specified primary efficacy measure was change from ture. The primary treatment group comparison was for the overall baseline to each postbaseline observation in UACR over the course of effect; however, the comparison at each time point was also per- the treatment period. The secondary efficacy measures included the formed. The secondary efficacy analysis for log UACR was an analysis proportion of subjects achieving at least a 40 and 25% reduction from of covariance of change from baseline to final observation. The model baseline in the last on-treatment UACR level and mean change in included the terms of treatment with baseline as the covariate. Similar estimated GFR. The same endpoints (for efficacy or safety) were used statistical models were applied to evaluate treatment group differ- to assess the changes 30 days after stopping treatment. ences in other continuous efficacy or safety variables, such as estimated UACR was assessed by a central laboratory using an immunotur- GFR, SBP, DBP, and hemoglobin. Treatment group differences in binary bidemetic method. Subjects were required to collect two consecutive random variables, such as the proportion of subjects who achieved at least first morning void urine samples before each scheduled visit. The 40% reduction on UACR and the incidence of treatment-emergent ad- geometric mean of the two samples was used as the UACR visit value verse events were evaluated using Fisher’s exact test. for subjects included in the analyses. Path analysis27 was used to test whether atrasentan 0.75 or 1.75 mg Pharmacokinetic Evaluation had a direct effect on UACR reduction after controlling for its thera- For all subjects in the study, blood samples for plasma atrasentan peutic effects of lowering SBP. Two regression models were used in ϭ ϩ ϫ ϩ concentrations were collected before dosing (0 hours), at 0.25, 1, 2 the path analysis: (1) change in log UACR a0 a1 treatment ϫ ϩ ϫ ϩ ϫ hours, and anytime between 4 and 6 hours after dosing on treatment a2 change in SBP a3 baseline in log UACR a4 baseline ϩ ϭ ϩ ϫ ϩ ϫ day 1. Additionally, for all subjects, one blood sample was collected SBP e1 and (2) change in SBP b0 b1 treatment b2 ϩ ϫ ϩ before dosing (0 hours) at treatment weeks 2, 4, 6, and 8 of the treat- baseline SBP b3 baseline log UACR e2. ment phase. In the analysis, a1 quantifies the direct effect and is tested by a t test; ϫ b1 a2 represents the indirect effect and its significance is tested by a Pharmacokinetic Variables Sobel’s t-ratio.28 The percentage of direct effect of the total effect (defined ϩ ϫ Atrasentan pharmacokinetic parameters including maximum plasma as a1 b1 a2) and the indirect effect of the total effect were calculated. concentration, time to maximum plasma concentration, and area un- The significance of the total effect can be tested by testing the treatment

770 Journal of the American Society of Nephrology J Am Soc Nephrol 22: 763–772, 2011 www.jasn.org CLINICAL RESEARCH

ϭ ϩ ϫ coefficient in a third regression model: change in log UACR c0 c1 Y, Oda N: The relationship between glycemic control and plasma ϩ ϫ ϩ ϫ vascular endothelial growth factor and endothelin-1 concentration in treatment c3 baseline of log UACR c4 baseline of SBP, when c ϭ a ϩ b ϫ a holds true. All analyses were performed using SAS diabetic patients. Metabolism 53: 550–555, 2004 1 1 1 2 7. Minchenko AG, Stevens MJ, White L, Abatan OI, Komjati K, Pacher P, version 9.1.3 (SAS Institute) under the Unix operating system. Szabo C, Obrosova IG: Diabetes-induced overexpression of endothelin-1 and endothelin receptors in the rat renal cortex is mediated via poly(ADP- ribose) polymerase activation. FASEB J 17: 1514–1516, 2003 ACKNOWLEDGMENTS 8. Kohan DE: Endothelin, hypertension and chronic kidney disease: New insights. Curr Opin Nephrol Hypertens 19: 134–139, 2010 9. Neuhofer W, Pittrow D: Endothelin receptor selectivity in chronic An abstract containing data from this study was submitted and ac- kidney disease: Rationale and review of recent evidence. Eur J Clin cepted for presentation at the American Society of Nephrology 2010 Invest 39[Suppl 2]: 50–67, 2009 meeting. This study was supported by Abbott Laboratories. We thank 10. Mann JF, Green D, Jamerson K, Ruilope LM, Kuranoff SJ, Littke T, Michael Amdahl, an Abbott statistician, for his contribution to the Viberti G: Avosentan for overt diabetic nephropathy. J Am Soc Neph- rol 21: 527–535, 2010 protocol design and Bo Yan, PhD, also an employee of Abbott, for his 11. Opgenorth TJ, Adler AL, Calzadilla SV, Chiou WJ, Dayton BD, Dixon DB, assistance with implementation of the statistical analysis plan. Medi- Gehrke LJ, Hernandez L, Magnuson SR, Marsh KC, Novosad EI, Von cal writing support was provided by Amanda Fein, PhD, on behalf of Geldern TW, Wessale JL, Winn M, Wu-Wong JR: Pharmacological char- Abbott. The sponsor was involved in the design of the study, in the acterization of A-127722: An orally active and highly potent ETA-selective collection and analysis of the data, and in writing the report. All au- receptor antagonist. J Pharmacol Exp Ther 276: 473–481, 1996 12. Luchner A, Hengstenberg C, Lowel H, Riegger GA, Schunkert H, thors had access to study results, and the lead author vouches for the Holmer S: Effect of compensated renal dysfunction on approved heart accuracy and completeness of the data reported. The lead author had failure markers: direct comparison of brain natriuretic peptide (BNP) the final decision to submit the publication. The study was overseen and N-terminal pro-BNP. Hypertension 46: 118–123, 2005 by a data review committee. The following investigators contributed 13. Kohan DE: Biology of endothelin receptors in the collecting duct. to subject recruitment for this study: Hanna Abboud, MD; Mario Kidney Int 76: 481–486, 2009 14. Ahn D, Ge Y, Stricklett PK, Gill P, Taylor D, Hughes AK, Yanagisawa M, Belledonne, MD; Diogo Belo, MD, FACP; Rafael Burgos-Calderon, Miller L, Nelson RD, Kohan DE: Collecting duct-specific knockout of MD; Jose L. Cangiano, MD; Douglas S. Denham, DO; Mohamed El- endothelin-1 causes hypertension and sodium retention. J Clin Invest Shahawy, MD, MPH, MHA; George Z. Fadda, MD; FACP; Venu M. 114: 504–511, 2004 Kondle, MD; K. Jean Lucas, MD; Robert G. Perry, MD; Edgardo Pi- 15. Ge Y, Bagnall A, Stricklett PK, Strait K, Webb DJ, Kotelevtsev Y, Kohan neiro, MD; Marc Rendell, MD; Jeffery Rosen, MD; Bhupinder Singh, DE: Collecting duct-specific knockout of the endothelin B receptor causes hypertension and sodium retention. Am J Physiol Renal Physiol MD, FASN; Mark Warren, MD, FACE; and Steven Zeig, MD. 291: F1274–F1280, 2006 16. Ge Y, Stricklett PK, Hughes AK, Yanagisawa M, Kohan DE: Collecting duct-specific knockout of the endothelin A receptor alters renal vaso- DISCLOSURES pressin responsiveness, but not sodium excretion or blood pressure. Am J Physiol Renal Physiol 289: F692–F698, 2005 D.K. is a consultant for Abbott Laboratories. M.M. is a consultant for Ab- 17. Wenzel RR, Littke T, Kuranoff S, Jurgens C, Bruck H, Ritz E, Philipp T, bott Laboratories and receives research support from Eli Lilly & Co., Ipsen, Mitchell A: Avosentan reduces albumin excretion in diabetics with Corcept, and Sanofi-Aventis. Y.P., S.W., P.A., and D.L.A. are employees of macroalbuminuria. J Am Soc Nephrol 20: 655–664, 2009 Abbott Laboratories, and Y.P., P.A., and D.L.A. own Abbott stock. 18. Shindo T, Kurihara H, Maemura K, Kurihara Y, Ueda O, Suzuki H, Kuwaki T, Ju KH, Wang Y, Ebihara A, Nishimatsu H, Moriyama N, Fukuda M, Akimoto Y, Hirano H, Morita H, Kumada M, Yazaki Y, Nagai R, Kimura K: Renal REFERENCES damage and salt-dependent hypertension in aged transgenic mice overex- pressing endothelin-1. J Mol Med 80: 105–116, 2002 19. Dhaun N, Ferro CJ, Davenport AP, Haynes WG, Goddard J, Webb DJ: 1. Gaede P, Lund-Andersen H, Parving HH, Pedersen O: Effect of a Haemodynamic and renal effects of endothelin receptor antagonism multifactorial intervention on mortality in type 2 diabetes. N Engl in patients with chronic kidney disease. Nephrol Dial Transplant 22: J Med 358: 580–591, 2008 3228–3234, 2007 2. Gilbertson DT, Liu J, Xue JL, Louis TA, Solid CA, Ebben JP, Collins AJ: 20. Sasser JM, Sullivan JC, Hobbs JL, Yamamoto T, Pollock DM, Carmines PK, Projecting the number of patients with end-stage renal disease in the Pollock JS: Endothelin A receptor blockade reduces diabetic renal injury via United States to the year 2015. J Am Soc Nephrol 16: 3736–3741, 2005 an anti-inflammatory mechanism. J Am Soc Nephrol 18: 143–154, 2007 3. Brenner BM, Cooper ME, de Zeeuw D, Keane WF, Mitch WE, Parving 21. Gagliardini E, Corna D, Zoja C, Sangalli F, Carrara F, Rossi M, Conti S, HH, Remuzzi G, Snapinn SM, Zhang Z, Shahinfar S: Effects of losartan Rottoli D, Longaretti L, Remuzzi A, Remuzzi G, Benigni A: Unlike each on renal and cardiovascular outcomes in patients with type 2 diabetes drug alone, lisinopril if combined with avosentan promotes regression and nephropathy. N Engl J Med 345: 861–869, 2001 of renal lesions in experimental diabetes. Am J Physiol Renal Physiol 4. Lewis EJ, Hunsicker LG, Clarke WR, Berl T, Pohl MA, Lewis JB, Ritz E, 297: F1448–F1456, 2009 Atkins RC, Rohde R, Raz I: Renoprotective effect of the angiotensin- 22. Battistini B, Berthiaume N, Kelland NF, Webb DJ, Kohan DE: Profile of receptor antagonist irbesartan in patients with nephropathy due to past and current clinical trials involving endothelin receptor antago- type 2 diabetes. N Engl J Med 345: 851–860, 2001 nists: The novel “-sentan” class of drug. Exp Biol Med (Maywood) 231: 5. Remuzzi G, Benigni A, Remuzzi A: Mechanisms of progression and 653–695, 2006 regression of renal lesions of chronic nephropathies and diabetes. 23. Raichlin E, Prasad A, Mathew V, Kent B, Holmes DR Jr, Pumper GM, J Clin Invest 116: 288–296, 2006 Nelson RE, Lerman LO, Lerman A: Efficacy and safety of atrasentan in 6. Kakizawa H, Itoh M, Itoh Y, Imamura S, Ishiwata Y, Matsumoto T, patients with cardiovascular risk and early atherosclerosis. Hyperten- Yamamoto K, Kato T, Ono Y, Nagata M, Hayakawa N, Suzuki A, Goto sion 52: 522–528, 2008

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24. de Zeeuw D, Remuzzi G, Parving HH, Keane WF, Zhang Z, Shahinfar 27. Lu Y: An application of path analysis in the design of clinical trials. Am S, Snapinn S, Cooper ME, Mitch WE, Brenner BM: Proteinuria, a Stat Assoc: 2576–2582, 2003 target for renoprotection in patients with type 2 diabetic nephrop- 28. Sobel, M: Asymptotic Confidence Intervals for Indirect Effects in Struc- athy: Lessons from RENAAL. Kidney Int 65: 2309–2320, 2004 tural Equation Models, San Francisco, Jossey-Bass, 1982 25. de Zeeuw D, Remuzzi G, Parving HH, Keane WF, Zhang Z, Shahinfar S, Snapinn S, Cooper ME, Mitch WE, Brenner BM: Albuminuria, a therapeutic target for cardiovascular protection in type 2 diabetic patients with nephropathy. Circulation 110: 921–927, 2004 See related editorial, “Endothelin Antagonist as Add-on Treatment for Protein- 26. Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D: A more uria in Diabetic Nephropathy: Is There Light at the End of the Tunnel?” on accurate method to estimate glomerular filtration rate from serum pages 593–595. creatinine: A new prediction equation. Modification of Diet in Renal Disease Study Group. Ann Intern Med 130: 461–470, 1999 Supplemental information for this article is available online at http://www.jasn.org/.

772 Journal of the American Society of Nephrology J Am Soc Nephrol 22: 763–772, 2011 Appendix 1

Supplementary Methods

Subject Inclusion Criteria and Exclusion Criteria: A subject was eligible for inclusion in the study if he/she met all of the following criteria: ≥18 years of age; voluntarily signed an informed consent form prior to any study specific procedures which was approved by an Institutional Review Board (IRB)/Independent Ethics Committee (IEC); type 2 diabetes and had been treated with ≥1 anti- hyperglycemic medication within the 12 months prior to Screening; receiving a stable dose of ACEi or ARB for ≥2 months prior to Screening; if female, not of childbearing potential (postmenopausal for ≥1 year of surgically sterile); and if male, surgically sterile or if sexually active and of childbearing potential with documented lack of desire for future procreation and agreement to use a condom and second reliable barrier of contraception from Screening until two months following completion of the study. In addition, subjects must have satisfied the following laboratory criteria during screening: eGFR >20mL/min/1.73m2 by simplified MDRD formula, UACR between 100 and 3000

mg/g, serum albumin >3.0 g/dL, HbA1c ≤10%, Pro-BNP ≤500 pg/mL (added as an addendum), and negative urine pregnancy test for female subjects.

A subject was excluded if he/she met the following criteria: history of significant peripheral edema, facial edema unrelated to trauma, or a history of myxedema in the 6 months prior to screening; received loop diuretics >30 mg BID of furosemide, >0.5 mg BID of bumetanide, or >25 mg BID of ethacrynic acid; history of pulmonary edema; history of pulmonary hypertension, chronic obstructive pulmonary disease, emphysema, pulmonary fibrous disease, asthma or other lung disease that required oxygen; documented history of heart failure (New York Heart Association Class II, III, or IV heart failure); body mass index >40; elevated liver enzymes (ALT and/or AST) >1.5 × the upper limit of normal; hemoglobin <9.5 g/dL; history of an allergic reaction or sensitivity to atrasentan or its excipients; history of a chronic gastrointestinal disease which in the Investigator’s opinion could cause significant gastrointestinal malabsorption; history of secondary hypertension; poorly controlled hypertension (systolic blood pressure ≥160 mm Hg or diastolic blood pressure ≥90 mm Hg) or hypotension (systolic blood pressure ≤90 mm Hg); comorbidities with a life expectancy of <1 year; subject was expected to receive an increased dose of RAS inhibitor (ACEi, ARB, renin or aldosterone inhibitor) during the course of the study (conversions from one product to another must have been equivalent doses); clinically significant coronary artery disease within 3 months prior to screening (hospitalization for myocardial infarction or unstable angina, new onset angina with positive functional study or coronary angiogram revealing stenosis, or coronary revascularization procedure); history of viral or bacterial infection within 4 weeks of Screening or HIV infection; surgery with general anesthesia within 12 weeks of Screening; history of drug or alcohol abuse within 6 months prior to Screening; evidence of poor compliance with diet or medication; received any investigational drug within 30 days prior to study drug administration; and if for any reason the subject was considered by the Investigator to be unsuitable to receive atrasentan orally or was put at risk by any study procedures.

Supplementary Figures

Figure 1. Atrasentan had no significant effect on body weight change.

Placebo 1.5 0.25 mg Atrasentan 0.75 mg Atrasentan 1.75 mg Atrasentan 1

0.5

Weight, kg Weight, -0.5

-1

-1.5 012345678 Time, Weeks

Figure 2. Effect of atrasentan on serum hemoglobin. All doses significantly reduced hemoglobin concentration which returned to baseline within 30 days of drug discontinuation.

Placebo 0.25 mg Atrasentan 0.75 mg Atrasentan 1.75 mg Atrasentan

0.1

-0.1

-0.3

-0.5

-0.7

-0.9 Hemoglobin, g/dL

-1.1

P<0.001 for any atrasentan vs. placebo -1.3 012345678930 Days Off Time, Weeks Figure 3. The effect of atrasentan on UACR reduction was present regardless of the level of RAS utilization.

60 Sub-maximal ACEi/ARB Maximum ACEi/ARB 50% 50

40 35% 32% 33% 30 27%

20 18% 19%

%UACR Reduction %UACR 10 0% 0 (n = 14) (n = 9) (n = 15) (n = 5) (n = 14) (n = 8) (n = 10) (n = 11) Placebo Atrasentan Atrasentan Atrasentan 0.25 mg 0.75 mg 1.75 mg