CTP 499 a Novel Drug for the Treatment of Chronic Kidney Disease CTP-499, a Novel Drug for the Treatment of Chronic Kidney Disease, CTP 499, a Novel Drug for the Treatment of Chronic Kidney Disease, Ameliorates Renal Fibrosis In Vivo and Modulates the IL 13 PathwayAmeliorates Renal Fibrosis In Vivo and Modulates the IL-13 PathwayAmeliorates Renal Fibrosis In Vivo and Modulates the IL 13 Pathway

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t hwww concertpharma com Kristine Hogan Kara West Gary W Bridson Lijun Wu PhD and Ara Aslanian PhD www.concertpharma.com Kristine Hogan, Kara West, Gary W Bridson, Lijun Wu, PhD and Ara Aslanian, PhD Le ington MA 02421

g , , y , j , ,C NCERT Ph ti l L i t M h tt U it d St t Lexington, MA 02421 CoNCERT Pharmaceuticals Lexington Massachusetts United States Lexington, MA 02421 CoNCERT Pharmaceuticals, Lexington, Massachusetts, United States.

CTP 499 i hibit kid fib i d t t i t t b l t i i th UUO d l CAbstract CTP-499 inhibits kidney fibrosis and protects against tubule apoptosis in the UUO model ConclusionsAbstract CTP 499 inhibits kidney fibrosis and protects against tubule apoptosis in the UUO model. ConclusionsRats were dosed with vehicle or CTP-499 (400 mg/kg BID, PO) 24 h before unilateral ureteral obstruction (UUO). Dosing continued for 10 days following surgery. Collagen accumulation was ( g g , ) ( ) g y g g y gmeasured by quantifying picosirius red stained kidney sections Mean plasma concentration of CTP-499 2 h after dosing = 10 9 M A Graph of average collagen volume fraction and measured by quantifying picosirius red stained kidney sections. Mean plasma concentration of CTP 499 2 h after dosing 10.9 M. A. Graph of average collagen volume fraction and representative sections shown below N = 8 for UUO groups N = 6 for sham ** p < 0 01 vs vehicle (t test) B H&E stained sections from UUO kidneys were scored for apoptotic tubule cells Chronic Kidney Disease (CKD) is a complex representative sections shown below. N = 8 for UUO groups, N = 6 for sham. ** p < 0.01 vs. vehicle (t-test). B. H&E-stained sections from UUO kidneys were scored for apoptotic tubule cells • CTP 499 is an investigational new Chronic Kidney Disease (CKD) is a complex, by a blinded observer. * p < 0.05 vs. vehicle (t-test). • CTP-499 is an investigational new

multifactorial disease in which renal function is y p ( ) g

drug for diabetic kidney disease that multifactorial disease in which renal function is i l i d R dl f drug for diabetic kidney disease that

10 BAprogressively compromised. Regardless of g yhas been ell tolerated in earl

10 2.5BAp g y p getiology decreased glomerular filtration rate due to has been well-tolerated in early BAetiology, decreased glomerular filtration rate due to y

li i l t di 8 ** 2.0

dysregulated fibrosis is a common feature of clinical studies. dysregulated fibrosis is a common feature of i CKD hi h l d d l

clinical studies. 6 1.5

progressive CKD which leads to end-stage renal progressive CKD which leads to end stage renal disease (ESRD) 4 1.0 *disease (ESRD). • In the rat UUO model CTP-499 ( ) In the rat UUO model, CTP-499

2 0.5

significantly reduced kidney fibrosis2 0.5

CTP-499 is a novel oral multi-subtype selective significantly reduced kidney fibrosis0 0 0CTP 499 is a novel oral multi subtype selective

h h di t (PDE) i hibit tl i in vivo the key pathological Sham Vehicle CTP-4990

Sham Vehicle CTP-4990.0

phosphodiesterase (PDE) inhibitor currently in a in vivo, the key pathological Sham Vehicle CTP 499 Sham Vehicle CTP 499p p ( ) yPhase II clinical trial for the treatment of CKD in

y gmechanism in diabetic kidney Phase II clinical trial for the treatment of CKD in mechanism in diabetic kidney

patients with type 2 diabetes While its full Representative pictures of picosirius red-stained sections from each treatment group Intense red stain represents areas of interstitial collagen accumulation area defined by computerized y

disease that leads to end stage renal patients with type 2 diabetes. While its full Representative pictures of picosirius red-stained sections from each treatment group. Intense red stain represents areas of interstitial collagen accumulation area defined by computerized l t ti disease that leads to end-stage renal

mechanism of action has yet to be elucidated we color segmentation. gdi mechanism of action has yet to be elucidated, we

h h th t CTP 499 i hibit i fl ti disease. have shown that CTP-499 inhibits inflammation, disease. ,reactive oxygen species generation mesangial cell reactive oxygen species generation, mesangial cell proliferation and epithelial-to-mesenchymal • Gene expression analysis of UUO proliferation and epithelial-to-mesenchymal Gene expression analysis of UUO transition all key pathophysiological processes that kidneys revealed that CTP-499 transition, all key pathophysiological processes that

t ib t t th i f fib i d CKDkidneys revealed that CTP-499

contribute to the progression of fibrosis and CKD. significantly reduced the expression p g significantly reduced the expression of IL 13 and downstream targets in In the current studies we demonstrate that CTP- of IL-13 and downstream targets in

Sh V hi l CTP 499In the current studies, we demonstrate that CTP- gUUO kidneys The IL 13/T 2 Sham Vehicle CTP-499

499 ameliorates renal fibrosis in vivo. Kidney UUO kidneys. The IL-13/TH2 S a e c e C 99499 ameliorates renal fibrosis in vivo. Kidney

ll t t d t b l ll t i y H

th i i t t di t f collagen content and tubule cell apoptosis was pathway is an important mediator of g p psignificantly reduced in rats dosed with CTP 499 CTP 499 reduces the expression of IL 13 and downstream targets of IL 13 (MCP 1 3 5 and

pathway is an important mediator of ti fib i ti th t significantly reduced in rats dosed with CTP-499 CTP-499 reduces the expression of IL-13 and downstream targets of IL-13 (MCP-1, 3, 5 and tissue fibrosis, suggesting that

compared to controls in the unilateral ureteral p g ( , ,

CCL22) d i i f IL 13R 2 i kidtissue fibrosis, suggesting that

compared to controls in the unilateral ureteral CCL22) and increases expression of IL-13R2 in kidney modulation of this pathway may be a obstruction (UUO) model. Also, gene array CCL22) and increases expression of IL 13R2 in kidney. modulation of this pathway may be a obstruction (UUO) model. Also, gene array l i l d th t i f IL 13 mRNA was isolated from UUO kidneys and genes were quantified by qPCR. * p < 0.05, ** p < 0.01 vs. vehicle (t-test). key anti-fibrotic mechanism of CTP-analysis revealed that expression of IL-13, a y g q y q p , p ( ) key anti-fibrotic mechanism of CTP-y p ,

profibrotic T 2 cytokine was upregulated in the 499 in vivo profibrotic TH2 cytokine, was upregulated in the 499 in vivo. obstructed kidneys of vehicle-treated rats CTP-obstructed kidneys of vehicle treated rats. CTP499 i ifi l i hibi d h i 499 treatment significantly inhibited the expression In vitro CTP 499 reduced the 499 treatment significantly inhibited the expression of IL 13 and other components of the IL 13/T 2 • In vitro, CTP-499 reduced the of IL-13 and other components of the IL-13/TH2 ,

lif ti f ti t d T ll p H

pathway including CCL22 a potent proliferation of activated T cells, a pathway including CCL22, a potent proliferation of activated T cells, a i f IL 13 i chemoattractant for TH2-polarized cells primary source of IL-13 expression chemoattractant for TH2 polarized cells. primary source of IL 13 expression

in vivo CTP-499 also inhibited To further explore this finding we investigated the

in vivo. CTP 499 also inhibited To further explore this finding, we investigated the secretion of IL-13 in response to p g geffects of CTP 499 on the IL 13/T 2 cell pathway in

secretion of IL-13 in response to effects of CTP-499 on the IL-13/TH2 cell pathway in stimulationvitro We found that CTP-499 inhibited IL-13 stimulation.vitro. We found that CTP 499 inhibited IL 13 ti i h i h l bl d l secretion in human peripheral blood mononuclear p p

cells (PBMC) and reduced proliferation of IL 13 has been shown to increase cells (PBMC) and reduced proliferation of • IL-13 has been shown to increase activated T-cells In addition we found that IL-13 CTP 499 reduces proliferation and IL 13 expression in stimulated T cells/PBMCs fib ti di t i i activated T-cells. In addition, we found that IL-13 CTP-499 reduces proliferation and IL-13 expression in stimulated T-cells/PBMCs. profibrotic mediators in various induces the expression of pro-fibrotic mediators in

p pHuman PBMCs were isolated by Ficoll gradient separation A Proliferation: PBMCs treated with CTP-499 were stimulated with PHA for 72 hours and proliferation was measured by BrdU

profibrotic mediators in various ti i l di l d li W induces the expression of pro fibrotic mediators in

kid i l ll d CTP 499 i ff ti t Human PBMCs were isolated by Ficoll gradient separation. A. Proliferation: PBMCs treated with CTP-499 were stimulated with PHA for 72 hours and proliferation was measured by BrdU ELISA M IC CTP 499 89 M f th i d d t i t B IL 13 ti PBMC t t d ith CTP 499 i t dditi f ti CD3/CD28 f 24 h S t d tissues including lung and liver. We kidney mesangial cells and CTP-499 is effective at ELISA. Mean IC50 CTP-499 = 89 M from three independent experiments. B. IL-13 secretion: PBMCs were treated with CTP-499 prior to addition of anti-CD3/CD28 for 24 hours. Secreted tissues including lung and liver. We y g

inhibiting this action IL-13 was quantified by ELISA. Mean IC50 CTP-499 = 186 M from two independent experiments. Representative data are shown. show that IL-13 induces expression inhibiting this action. q y 50 p p p show that IL 13 induces expression of CTGF and PAI-1 in kidney 600120 BA of CTGF and PAI-1 in kidney BATaken together, these data show that CTP-499 mesangial cells and that CTP-499 500100 BATaken together, these data show that CTP 499

ti fib ti d t ti mesangial cells and that CTP-499

40080possesses anti-fibrotic and renoprotective can inhibit this effect 40080p pproperties in vivo Inhibition of IL 13 expression can inhibit this effect.

30060properties in vivo. Inhibition of IL-13 expression 30060

and activity in combination with other disease- 20040and activity in combination with other disease-CTP 499 i h h di t

20040

modifying activities described for PDE inhibitors • CTP-499 is a phosphodiesterase 10020modifying activities described for PDE inhibitors t ib t t th ti fib ti h i f

C 99 s a p osp od este ase(PDE) i hibit ith ifi it f

100

may contribute to the anti-fibrotic mechanism of (PDE) inhibitor with specificity for 00yCTP 499 These results further support our

(PDE) inhibitor with specificity for l b i l di PDE3 4

-7 -6 -5 -4 -3 -2-7 -6 -5 -4 -3 -2CTP-499. These results further support our select subtypes including PDE3 4 Log [CTP-499] MLog [CTP-499] Mcontinued interest in CTP-499 as a novel agent for select subtypes including PDE3, 4 Log [CTP 499], MLog [CTP 499], Mcontinued interest in CTP 499 as a novel agent for h i l f CKD and 5 The scope of biological the potential treatment of CKD. CTP 499 i hibit IL 13 i d d i f fib ti di t d h ki f kid

and 5. The scope of biological the potential treatment of CKD. CTP-499 inhibits IL-13-induced expression of pro-fibrotic mediators and chemokines from kidney effects observed in vivo and in vitro CTP 499 inhibits IL 13 induced expression of pro fibrotic mediators and chemokines from kidney effects observed in vivo and in vitro mesangial cells with CTP 499 are consistent with mesangial cells. with CTP-499 are consistent with gRat mesangial MC-1 cells were treated with IL-13 and CTP-499 as indicated for 6 hours Gene expression analysis was performed and quantified by qPCR GAPDH was used as an internal this activityRat mesangial MC-1 cells were treated with IL-13 and CTP-499 as indicated for 6 hours. Gene expression analysis was performed and quantified by qPCR. GAPDH was used as an internal control * p < 0 05 ** p <0 01 *** p <0 001 **** p <0 0001 s IL 13 treated cells (t test) IL 13 treatment gro p p al e is relati e to n stim lated cells this activity.control. * p < 0.05, ** p <0.01, *** p <0.001, **** p <0.0001 vs. IL-13 treated cells (t-test). IL-13 treatment group p value is relative to un-stimulated cells y

3 ******

Structure of CTP 499Structure of CTP-499 2 **Structure of CTP 499CTP-499, is a novel, deuterated analog of 1-((S)-5- 1 ***CTP 499, is a novel, deuterated analog of 1 ((S) 5hydroxyhexyl) 3 7 dimethylxanthine (HDX)

1

hydroxyhexyl)-3,7-dimethylxanthine (HDX)0

F f th i f ti- - 30 M 100 M 300 M

0CTP-499 For further informationIL-13 - + + + + For further information

Ara AslanianAra AslanianDirector PharmacologyDirector, Pharmacology

l i @ t haaslanian@concertpharma.com@ p

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