CTP 499 a Novel Drug for the Treatment of Chronic Kidney Disease ... [PDF]

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

www.concertpharma.com www concertpharma t h com Le ington MA 02421 Lexington,

Abstract Chronic Kidney Disease (CKD) is a complex, complex multifactorial disease in which renal function is pprogressively g i ly compromised. p i d R Regardless g dl off etiology decreased glomerular filtration rate due to etiology, dysregulated fibrosis is a common feature of progressive i CKD which hi h lleads d to end end-stage d stage renall disease (ESRD) ((ESRD). ) CTP 499 is a novel oral multi CTP-499 multi-subtype subtype selective pphosphodiesterase h ph di t (PDE) ( ) inhibitor i hibit currently tly in i a Phase II clinical trial for the treatment of CKD in patients with type 2 diabetes. diabetes While its full mechanism of action has yet to be elucidated, elucidated we h have shown h that th t CTP-499 CTP 499 iinhibits hibit inflammation, i fl ti , reactive oxygen species generation, generation mesangial cell proliferation and epithelial-to-mesenchymal transition all key pathophysiological processes that transition, contribute t ib t to t the th progression p g i off fibrosis fib i andd CKD. CKD In the current studies studies, we demonstrate that CTP499 ameliorates renal fibrosis in vivo. Kidney collagen ll g content t t andd tubule t b l cellll apoptosis p pt i was significantly reduced in rats dosed with CTP-499 CTP 499 compared to controls in the unilateral ureteral obstruction (UUO) model. Also, gene array analysis ly i revealed l d that th t expression p i off IL-13, IL 13, a profibrotic TH2 cytokine, cytokine was upregulated in the obstructed kidneys of vehicle vehicle-treated treated rats rats. CTPCTP 499 treatment significantly i ifi l inhibited i hibi d the h expression i of IL-13 IL 13 and other components p of the IL-13/T IL 13/TH2 pathway including CCL22 CCL22, a potent chemoattractant for TH22-polarized polarized cells. cells To further explore p this finding, findingg we investigated g the effects of CTP-499 CTP 499 on the IL-13/T IL 13/TH2 cell pathway in vitro We found that CTP vitro. CTP-499 499 inhibited IL IL-13 13 secretion ti iin hhuman pperipheral iph l blood bl d mononuclear l cells (PBMC) and reduced proliferation of activated T-cells. T-cells In addition, addition we found that IL-13 induces the expression of pro pro-fibrotic fibrotic mediators in kid y mesangial kidney gi l cells ll andd CTP-499 CTP 499 is i effective ff ti att inhibiting this action. action Taken together, these data show that CTP CTP-499 499 ppossesses anti-fibrotic ti fib ti andd renoprotective p t ti properties in vivo. vivo Inhibition of IL IL-13 13 expression and activity in combination with other diseasemodifying activities described for PDE inhibitors mayy contribute t ib t to t the th anti-fibrotic ti fib ti mechanism h i off CTP 499 These results further support our CTP-499. continued interest in CTP CTP-499 499 as a novel agent for the h potential i l treatment off CKD. CKD

Kristine Hogan, Hogan g , Kara West, West, Garyy W Bridson, Bridson, Lijun j Wu, Wu, PhD and Ara Aslanian, Aslanian, PhD C NCERT Pharmaceuticals, CoNCERT Ph Pharmaceuticals ti l Lexington, Lexington L i t Massachusetts, Massachusetts M h tt United U it d States. States St t CTP-499 CTP 499 iinhibits hibit kid kidney fib fibrosis i andd protects t t against i t tubule t b l apoptosis t i in i the th UUO model. model d l Rats were dosed with vehicle or CTP-499 (400 ( mg/kg g g BID,, PO)) 24 h before unilateral ureteral obstruction (UUO). ( ) Dosingg continued for 10 days y followingg surgery. g y Collagen g accumulation was measured by quantifying picosirius red stained kidney sections. sections Mean plasma concentration of CTP CTP-499 499 2 h after dosing = 10.9 10 9 M. M A. A Graph of average collagen volume fraction and representative sections shown below. below N = 8 for UUO groups groups, N = 6 for sham. sham ** p < 0.01 0 01 vs. vs vehicle (t-test). (t test) B. B H&E-stained H&E stained sections from UUO kidneys were scored for apoptotic tubule cells byy a blinded observer. * p < 0.05 vs. vehicle ((t-test). )

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CTP-499 CTP 499 is an investigational g new drug g for diabetic kidneyy disease that has been well-tolerated ell tolerated in earl earlyy clinical li i l studies. t di

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In the rat UUO model model, CTP-499 significantly reduced kidney fibrosis in vivo, vivo the keyy pathological g mechanism in diabetic kidneyy disease that leads to end-stage end stage g renal di disease.

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Gene expression analysis of UUO kidneys revealed that CTP-499 significantly reduced the expression of IL-13 IL 13 and downstream targets g in UUO kidneys kidneys. y The IL IL-13/T 13/TH2 pathway th iis an iimportant t t mediator di t off ti tissue fibrosis, fib i suggesting ti th thatt modulation of this pathway may be a key anti-fibrotic mechanism of CTP499 in vivo. vivo

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In vitro, vitro, CTP-499 CTP 499 reduced the proliferation lif ti off activated ti t d T cells, ll a primary i source off IL IL-13 13 expression i in vivo vivo. CTP CTP-499 499 also inhibited secretion of IL-13 in response to stimulation stimulation.

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IL-13 IL 13 has been shown to increase profibrotic fib ti mediators di t in i various i ti tissues iincluding l di llung and d liliver. W We show that IL IL-13 13 induces expression of CTGF and PAI-1 in kidney mesangial cells and that CTP-499 can inhibit this effect. effect

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C 499 CTP CTP-499 99 is is a phosphodiesterase phosphod dieste t ase (PDE) iinhibitor hibit with ith specificity ifi it for f select l subtypes b iincluding l di PDE3 PDE3, 4 and 5. 5 The scope of biological effects observed in vivo and in vitro with CTP-499 CTP 499 are consistent with this activity. activityy

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Representative pictures of picosirius red-stained sections from each treatment group. group Intense red stain represents areas of interstitial collagen accumulation area defined by computerized color l segmentation. t ti

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CTP-499 CTP 499 reduces the expression p of IL-13 IL 13 and downstream targets g of IL-13 IL 13 (MCP-1, (MCP ( 1, 33,, 5 and CCL22) and d iincreases expression i off IL IL-13R2 13R 2 iin kidney. 13R2 kidney kid mRNA was isolated from UUO kidneys y and ggenes were qquantified byy qqPCR. * p < 0.05,, ** p < 0.01 vs. vehicle ((t-test). )

CTP 499 reduces pproliferation and IL-13 CTP-499 IL 13 expression p in stimulated T-cells/PBMCs. T cells/PBMCs Human PBMCs were isolated by Ficoll gradient separation separation. A A. Proliferation: PBMCs treated with CTP-499 were stimulated with PHA for 72 hours and proliferation was measured by BrdU ELISA Mean ELISA. M IC50 CTP-499 CTP 499 = 89 M M ffrom th three iindependent d d t experiments. i t B. B IL-13 IL 13 secretion: ti PBMCs PBMC were treated t t d with ith CTP-499 CTP 499 prior i to t addition dditi off anti-CD3/CD28 ti CD3/CD28 ffor 24 hhours. Secreted S t d IL-13 was qquantified byy ELISA. Mean IC50 CTP-499 = 186  M from two independent p experiments. p Representative p data are shown.

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CTP 499 inhibits CTP-499 i hibit IL IL-13-induced 13 induced i d d expression i off pro pro-fibrotic fib ti mediators fibrotic di t andd chemokines h ki from f kidney kid mesangial g cells. cells Rat mesangial MC-1 cells were treated with IL-13 and CTP-499 as indicated for 6 hours. hours Gene expression analysis was performed and quantified by qPCR. qPCR GAPDH was used as an internal control * p < 0.05, control. 0 05 ** p