Lesinurad (RDEA594), A Novel Oral Uricosuric Agent, in Combination with Febuxostat Shows Significant Additive
Urate Lowering Effects in Gout Subjects with 100% Response Achieved for All Combination Dose Regimens
R. Fleischmann¹, Z. Shen², L. Yeh², B. Kerr², E. Polvent², M. Suster², V. Hingorani², J.N. Miner², K. Manhard², B. Quart^2
1Metroplex Clinical Research Center, Dallas, ²Ardea Biosciences Inc, San Diego, CA

Introduction

Lesinurad (RDEA594) is a uricosuric agent in development for the treatment of gout, which acts through inhibition of uric acid transporters (URAT1 and OAT4 — EULAR2011—THU0025) in the proximal tubule of the kidney. Lesinurad has been well tolerated, with dose-dependent reductions in serum urate (sUA) in over 500 subjects who have received lesinurad in Phase 1 & 2 clinical trials. Febuxostat (Adenuric^®, Uloric^®, FBX) is a xanthine oxidase (XO) inhibitor recently approved in the EU (80 mg -120 mg once daily (QD) and the US (40 mg — 80 mg QD). In a published study, an increased speed of tophi reduction was observed when XO and URAT1 inhibitors were given in combination¹. The studies described below were designed to evaluate the benefit of combining two mechanisms of sUA lowering as illustrated in Figure 1. This dual mechanism approach with lesinurad is expected to produce target sUA reductions (response is defined as sUA < 6 mg/dL) in gout patients and may ideally be suited to patients with high sUAs and tophaceous gout. Simultaneously targeting these two different mechanisms for sUA lowering has produced significantly increased reductions in sUA over either agent alone, providing the potential for faster tophi resolution and a decrease in the rate of gout flares with chronic treatment.^1,2

Figure 1. Dual Mechanism of sUA Reduction: Decreasing Urate Production by XO Inhibition and Increasing Urate Excretion by URAT1 Inhibition Leads to Greater sUA Lowering

In a prior drug-drug interaction study in healthy volunteers, lesinurad 200 mg and 400 mg in combination with FBX 40 mg showed no clinically relevant pharmacokinetic (PK) drug-drug interaction between lesinurad and FBX. Lesinurad with a low dose of FBX produced substantial additive effects on sUA in these subjects, with reductions of up to ~70%. The combination of lesinurad with FBX was well tolerated.

Methods

RDEA594-111 was a multicenter, open-label, multiple-dose study in subjects with gout designed to evaluate the sUA lowering effects, PK, drug-drug interaction potential, safety and tolerability of lesinurad in combination with FBX (Figure 2). The study included 21 subjects in 2 panels. All subjects initially received FBX alone for 7 days, followed by combination treatment with lesinurad 400 mg QD for 7 days, and then combination treatment with lesinurad 600 mg QD for 7 days.

Figure 2. Study design for RDEA594-111

Key Inclusion Criteria:

Ø		Healthy adult males or post-menopausal/surgically sterile females ³ 18 and £ 80 years of age
Ø		Met one or more criteria for the diagnosis of gout as per the ARA Criteria for the Classification of Acute Arthritis of Primary Gout
Ø		sUA level ³ 8 mg/dL
Ø		Body mass index of £ 40 kg/m2
Ø		All laboratory parameters within normal limits or not clinically significant

Assessments:

Ø		Adverse events (AEs), clinical laboratory test results, vital signs, 12—lead electrocardiograms (ECGs), and physical examination for safety
Ø		PK of lesinurad and of FBX in plasma
Ø		Pharmacodynamics including sUA lowering and urine urate excretion

Objectives:

Ø

Primary

	•		To evaluate the pharmacokinetics of FBX alone and in combination with lesinurad
	•		To evaluate the pharmacokinetics and urinary excretion of lesinurad in combination with FBX
	•		To evaluate the pharmacokinetics of colchicine alone and in combination with FBX or both febuxostat and lesinurad.

Ø Secondary

	•		To measure the effect of FBX alone and in combination with lesinurad on serum urate concentrations and urinary urate excretion
	•		To evaluate the safety and tolerability of multiple doses of lesinurad in combination with FBX

Results

Disposition and Demographics

Twenty-one subjects enrolled and 20 completed the study. One subject in Panel 1 was discontinued on Day 14 due to a protocol violation (positive drug screen).

Table 1. Subject Demographics

		Panel 1 (FBX 40 mg)				Panel 2 (FBX 80 mg)
Baseline Variable		(N=12)				(N=9)
Age, years [Mean (SD)]			52 (14.3	)			43 (11.1	)
Body weight, kg [Mean (SD)]			101.6 (24.44	)			105.5 (19.03	)
BMI, kg/m2 [Mean (SD)]			32.8 (5.82	)			32.1 (5.06	)
sUA, mg/dL (Median)			9.2				10.4
Race [n (%)]
Asian			1 (8	%)			0
Black			4 (33	%)			3 (33	%)
White			7 (58	%)			6 (67	%)
Ethnicity [n (%)]
Not Hispanic or Latino			12 (100	%)			9 (100	%)

Results (cont’d)

Pharmacodynamics

Table 2.

Combination of Lesinurad + FBX Resulted in Significant sUA Reduction by both Median Percent and Absolute Change at Trough and Peak

										Trough												12-hrs post-dose
FBX										FBX												FBX
dose														+ lesinurad				+ lesinurad								+ lesinurad				+ lesinurad
(mg)				sUA Variable		Baseline				Alone				400 mg				600 mg				Alone				400 mg				600 mg
	40			sUA levels (mg/dL)			9.2				5.7				3.9				3.8				5.2				2.7				2.4
	(n=12	)		Absolute reduction (mg/dL)			—				-3.2				-5.4				-5.8				-3.9				-6.5				-6.8
				Percent change (%)			—				-35	%			-56	%			-61	%			-44	%			-68	%			-71	%
	80			sUA levels (mg/dL)			10.4				5.8				3.3				2.8				4.8				2.3				2.0
	(n=9	)		Absolute reduction (mg/dL)			—				-4.2				-6.7				-7.3				-5.1				-8.0				-8.4
				Percent change (%)			—				-47	%			-65	%			-73	%			-52	%			-78	%			-81	%

Figure 3. Combination of Lesinurad + FBX Significantly Reduced sUA Compared to FBX Alone

Ø		Clinically important reductions in sUA of up to 80% were observed in gout subjects with the combination of lesinurad and FBX
Ø		With lesinurad 400 mg dose in panels 1 and 2, subjects achieved median sUA levels of 3.9 mg/dL and 3.3 mg/dL at trough, respectively; and 2.7 mg/dL and 2.3 mg/ dL, respectively, at 12-hrs post-dose
Ø		With lesinurad 600 mg dose in panels 1 and 2, subjects achieved median sUA levels of 3.8 mg/dL and 2.8 mg/dL at trough, respectively; and 2.4 mg/dL and 2.0 mg/ dL, respectively, at 12-hrs post-dose

Figure 4. All Subjects Achieved a Clinical Response (sUA < 6 mg/dL) in all Combination Regimens of Lesinurad + FBX at Trough

Ø

At trough;

	•		100% of subjects receiving the combination of lesinurad and FBX achieved sUA levels below the clinically important target of 6 mg/dL, compared to 67% and 56% for 40 mg and 80 mg, respectively, with FBX alone
	•		75 to 100% of subjects receiving the combination of lesinurad and FBX achieved sUA levels below of 5 mg/dL, compared to 17% and 22% for 40 mg and 80 mg, respectively, with FBX alone. Reduction of sUA below 5 mg/dL may be clinically important for subjects with tophi, as the velocity of tophi resolution has been shown to be enhanced with lower sUA 1

Figure 5. Over 90% of Subjects Achieved sUA < 4 mg/dL in all Combination Regimens of Lesinurad + FBX at 12-hrs Post-Dose

Ø

At 12-hrs post-dose;

	•		Even greater responses were seen with combination therapy compared to FBX alone.
	•		100% of subjects achieved sUA levels below 4 mg/dL on lesinurad 400 mg or 600 mg combined with 80 mg FBX, and 100% of subjects achieved levels below 3 mg/dL on lesinurad 600 mg combined with 80 mg FBX. No subject achieved these reduced sUA levels on either dose of FBX alone. As noted above, lower sUA is associated with faster resolution of tophi in a study by Perez-Ruiz.1 Increased resolution of tophi was also associated with lower sUA in Phase 3 trials of pegloticase, where the regimen producing a mean plasma urate of approximately 3 mg/dL produced the greatest tophi response3
	•		The combination of FBX 40 mg plus lesinurad 400 mg was significantly superior to the 80 mg dose of FBX alone (80 mg/day is the highest approved dose in the US)

Table 3.

The Combination of Lesinurad + FBX should not Increase Risk for Kidney Stones based on 24 hr Undissociated Urine Urate Levels3 and Urine pH

Mean (SE) Undissociated 24h Urine Urate Concentration in mg/dL

						FBX
Panel		Baseline				Alone				+ Lesinurad 400 mg				+ Lesinurad 600 mg
FBX 40 mg			2.7 (0.6	)			1.6 (0.4	)			1.9 (0.5	)			2.3 (0.7	)
FBX 80 mg			11 (1.8	)			3.9 (1.0	)			4.8 (1.6	)			2.3 (0.6	)
Mean (SE) 24h Urine pH
FBX 40 mg			5.9 (0.1	)			5.9 (0.1	)			6.0 (0.1	)			6.0 (0.1	)
FBX 80 mg			5.5 (0.1	)			5.6 (0.1	)			5.7 (0.2	)			5.8 (0.1	)

Ø		Mean (SE) undissociated urine urate levels (mg/dL) remained well below 10 mg/dL, which decreases the risk of uric acid stones 4
Ø		Urine pH increased or remained unchanged in gout subjects

Pharmacokinetics

Table 4. Steady State Plasma FBX Exposures were Mildly Increased by Lesinurad

										Geometric Mean Ratio% (CI90%)
Dose (mg)										(Combo/Alone)
FBX		Lesinurad				N				Cmax				AUC
40			400				11				109 (83-143	)			108 (99-117	)
			600				11				129 (109-154	)			120 (109-132	)
80			400				9				113 (104-123	)			119 (112-126	)
			600				9				118 (93-148	)			121 (107-137	)

Ø		In gout subjects, a mild increase (< 30%) in FBX exposure was seen with co-administration with lesinurad
Ø		Lesinurad exposure was not altered by FBX

Safety

Ø		Overall, the combination of lesinurad and FBX was well tolerated, with no serious adverse events, no discontinuations due to adverse events, and no increase in adverse events with the combination
Ø		No difference in rate of adverse events across dosing periods, with lowest rate observed in the lesinurad 600 mg + FBX arms (Table 5)
Ø		Renal safety: 1 subject had a grade 2 increase in serum creatinine (SCr) on colchicine alone; no subjects with grade 2 or greater SCr increases on lesinurad or the combination
Ø		Liver safety: 1 subject had a grade 3 increase in ALT on FBX 40 mg alone, which normalized during combination treatment
Ø		12 subjects had at least one CK elevation above the normal range; however, 8 of those subjects had elevated CKs at screening and/or baseline. Of the remaining 4 subjects, 2 subjects had 1 elevation each, 1 subject had 2 elevations, and 1 subject had 4 elevations. None of the elevations were considered by the investigator to be clinically significant

Table 5. Treatment-emergent Adverse Events Reported in 2 or More Subjects

		40 mg FBX												80 mg FBX
						+ Lesinurad				+ Lesinurad								+ Lesinurad				+ Lesinurad
MedDRA Preferred		_				400 mg				600 mg				_				400 mg				600 mg
Term		(N=12)				(N=12)				(N=11)				(N=9)				(N=9)				(N=9)
Dyspepsia			0				1 (8	%)			0				0				2 (22	%)			0
Gout Flares			2 (17	%)			0				0				0				1 (11	%)			3 (33	%)
Headache			1 (8	%)			1 (8	%)			0				0				3 (33	%)			0

Conclusions

Ø		In gout subjects, the combination of lesinurad and FBX produced substantial additive sUA lowering with 100% response rate (< 6 mg/dL) achieved at all combination dose levels, 100% of subjects achieved < 4 mg/dL at highest doses at trough, and 100% of subjects achieved sUA < 3 mg/dL intraday.
Ø		Combining drugs with complementary mechanisms produced significantly greater reductions in sUA than increasing the dose of a single agent.
Ø		No clinically relevant pharmacokinetic drug-drug interactions between lesinurad and FBX were noted in gout subjects at the doses tested.
Ø		Given that mono-sodium urate crystals are soluble below 6.8 mg/dL, the ability of the combination of lesinurad and FBX to bring sUA in 100% of subjects to < 3 mg/dL may be particularly useful in subjects who have accumulated large deposits of uric acid, or tophi.
Ø		The combination was generally well tolerated with no deaths or other serious adverse events and no subjects discontinued from the study due to adverse events.
Ø		Multiple QD oral doses of 400 and 600 mg lesinurad were generally safe and well tolerated by gout subjects with hyperuricemia when administered in combination with 40 and 80 mg FBX QD.

Reference:

1.		Perez-Ruiz F, Arthritis Rheum 2002;47:356-360.
2.		Becker MA, et al. 2007 ACR Annual Meeting, Abstract No. 758.
3.		FDA Advisory Committee Briefing Book for Pegloticase
		Asplin J, Nephrology, 1996; 16: 412-424

Annual European Congress of Rheumatology EULAR, London, May 25 — 28, 2011

THU0025

Lesinurad (RDEA594), A Investigational Uricosuric Agent for Hyperuricemia and Gout,
Blocks OAT4 Transport, Mechanism of Hydrochlorothiazide-Dependent Hyperuricemia
P.K. Tan, D. Hyndman, S. Liu, B. Quart and J.N. Miner,
ARDEA BIOSCIENCES, San Diego, CA, United States

Introduction

Lesinurad (RDEA594) is a urate lowering therapy in clinical development for the treatment of gout that blocks reabsorption of urate (UA) within the kidney proximal tubule by inhibiting the URAT1 transporter. Lesinurad shows significant urate lowering activity in both monotherapy and in combination with xanthine oxidase inhibitors. In addition to URAT1, organic anion transporter 4 (OAT4) is also considered an important regulator of urate excretion. As such, lesinurad was tested to determine its activity against OAT4 and was found to be active against OAT4 at concentrations that would be observed at the site of action. Activity against OAT4 may provide important additional pharmacologic properties, as it has been postulated that OAT4 may be responsible for the hyperuricemia associated with some diuretics. Hypertension is prevalent in gout patients and they are often treated with anti-hypertensive agents, such as thiazide diuretics, which have been known since the 1950’s to elevate serum UA levels¹. Urate lowering therapies are generally believed to work less efficiently in patients taking diuretics concomitantly². This effect is thought to be mediated by enhanced UA reabsorption due to activation of OAT4 by diuretics via two different mechanisms^3,4.

Figure 1. Two mechanisms for HCTZ-induced hyperuricemia

•		Direct -HCTZ enhancement of Uric acid uptake by OAT42
•		Indirect -HCTZ enhances an OAT4 stimulatory protein (NHE3)3

Data presented with permission Johannes Hagos

Methods

The UA transporter OAT4 was stably expressed in cultured cells and oocytes. For cultured cells, HEK293 cells stably expressing the transporters were produced through transfection of DNA constructs carrying the transporters, antibiotic selection, and clonal selection of clones with high transporter activity. Alternatively, cells transiently expressing the transporters were produced by reverse transfection of HEK293T cells. Transfectants were plated at high density onto poly-L-lysine coated multiwell plates and assayed 1-2 days later. Results were similar for stable and transient expressing cells. Oocytes were injected with cRNA for OAT4 expression and then assayed 3-4 days later.

Activity assays were performed by incubating the cells with transporter substrates in assay buffer containing 125 mM sodium gluconate, 4.8 mM potassium gluconate, 1.2 mM monobasic sodium phosphate, 1.2 mM magnesium sulfate, 1.3 mM calcium gluconate, 5.6 mM glucose, and 25 mM HEPES pH 7.1. Drugs such as lesinurad were added to the cells prior to addition of substrate for the indicated times. Substrates used were 6-carboxyfluorescein (CF) at 5 µM, 3H-estrone sulfate (ES) at 50 nM, and 14C-uric acid (UA) at 100 µM. For transfected cells, substrates were incubated for 2 minutes and then removed by aspiration and the cells washed three times in a wash buffer containing 125 mM sodium gluconate and 25 mM HEPES pH 7.1. Cells were then lysed in 1 M sodium hydroxide prior to fluorescence measurement for CF transport and liquid scintillation counting for ES and UA. Oocyte assays were performed similarly, except that drugs such as lesinurad were injected and then transport was measured after 30 minutes (ES).

Results

Figure 2. Lesinurad inhibits OAT4 transport activity

Figure 3. Urate acts as a competitive substrate for OAT4 of CF

Human Kidney cells (HEK293) exhibit OAT4-dependent transport of CF that is blocked by lesinurad.

293T cells transiently transfected with OAT4 or control plasmid lacking OAT4 (pCMV) were incubated with CF, with vehicle (green) or 50 uM Lesinurad (red).

EC50 ~900 uM

293T cells transiently transfected with OAT4 blue or control plasmid lacking OAT4 (pCMV, black) were incubated with CF without or with different amounts of cold UA.

Figure 4. Lesinurad inhibits urate transport activity by OAT4

Lesinurad and benzbromarone are inhibitors of OAT4, with IC₅₀’s of 5 and 10 µM, respectively. Concentrations of compound likely found in proximal tubule are shown shaded (red —lesinurad, grey — benzbromarone).

Figure 5. Lesinurad inhibits URAT1 and OAT4 with similar potency

UA transport was measured in 293T cells transiently expressing URAT1 or 293 cells stably expressing OAT4. Inhibition was measured after a 5 minute incubation in the indicated concentrations of lesinurad.

Figure 6. HCTZ has no effect on lesinurad-mediated inhibition of OAT4 transporter activity

293 cells stably expressing OAT4 were incubated with 3H-Estrone sulphate for 5 min in the presence or absence of 1 mM HCTZ in the media. A dose response was conducted with lesinurad and percent transported ES was measured and graphed.

Figure 7. Oxypurinol and allopurinol do not inhibit OAT4 transport activity

100 µM of lesinurad, oxypurinol, or allopurinol was incubated for 5 minutes with OAT4-expressing 293 cells prior to addition of OAT4 substrates 14C-UA (A) or CF (B).

Figure 8A. Lesinurad likely inhibits OAT4 from the extracellular (apical) side

Oat4-expressing oocytes were injected with 50 nl of various concentrations of lesinurad or vehicle intracellularly (inside) and uptake of 3H Estrone Sulfate was measured. For extracellular inhibition (outside) lesinurad or vehicle was added directly to media at the indicated concentrations.

Figure 8B. Control experiment demonstrating that lesinurad remains inside injected oocytes for the duration of the experiment

Oat4-expressing oocytes were injected with 50 nl of ¹⁴C-labeled lesinurad in water. ¹⁴C was measured in 8 Oocytes immediately (T=0). Remaining oocytes were incubated for 30 minutes, then ¹⁴C levels were measured in the buffer (outside). In addition, the amount of ¹⁴C remaining in the oocytes was measured (inside).

Figure 9. Phase 2 Study 203 — Design

Study 203, a 4-week, double-blind, placebo-controlled trial of lesinurad in 208 gout patients who were not adequately responding to allopurinol with serum urate (sUA) ³ 6 mg/dL while receiving a stable dose of allopurinol for at least 6 weeks. One of three doses of lesinurad or matching placebo was added to the patients allopurinol regimen. Primary endpoint was mean reduction in sUA at Week 4, with the key secondary endpoint the proportion of subjects with sUA < 6.0 mg/dL at Week 4. A small number of patients in this trial received a thiazide diuretic during the treatment period; their response rates were compared to those patients not receiving concomitant diuretics.

Figure 10. Proportion of patients responding in Study 203. Patients receiving diuretics had an excellent response to lesinurad

Results (cont’d)

Conclusions

Ø		The ability of OAT4 to transport urate from the urine side to the blood side is enhanced by thiazide diuretics, which are known to produce hyperuricemia
Ø		Lesinurad inhibits OAT4 from the apical (urine) side in the proximal tubule
Ø		Lesinurad inhibits OAT4 with similar potency as URAT1 at concentrations achieved in treated patients suggesting that lesinurad could lower sUA in patients receiving thiazide diuretics better than both xanthine oxidase inhibitors and other URAT1 inhibitors
Ø		Since thiazide diuretics are one of the most common treatments for hypertension, this additional activity could be beneficial for many patients
Ø		Despite small numbers of patients taking thiazide diuretics in the 203 study, the high response to lesinurad in diuretic-treated patients is consistent with OAT4 inhibition by lesinurad
Ø		The activity of lesinurad in patients receiving thiazide diuretics will be studied further during Phase 3

References

1.		Healey et al, NEJM 1959; 261:1358-1362
2.		Reyes AJ. Cardiovasc Drugs Ther. 2003 Sept-Nov; 17(5-6): 397-414.
3.		Hagos et al, J Am Soc Nephrol 18: 430, 2007
4.		Nijenhuis T, Vallon V, van der Kemp AW, Loffing J, Hoenderop JG, Bindels RJ. J Clin Invest 2005, 115: 1651-1658.

     Annual European Congress of Rheumatology EULAR, London, May 25 — 28, 2011