Development of Novel Therapeutics for Glaucoma UMN-Mayo Partnership Project Update

Overview • Glaucoma – leading cause of irreversible blindness • Causal and most prevalent risk factor is elevated IOP • All treatments are aimed at reducing IOP • ATP-sensitive potassium channel openers • Lower IOP in ex vivo and in vivo models • Commercial compounds are not water-soluble

• Development of soluble prodrugs • Cromakalim was conducive to chemical modifcication

• CKLP1 is lead compound • Water solubility • Lowers IOP in ex vivo model and in vivo in mice

• Provisional patent filed January 2014 (Page 2)

ATP sensitive potassium (KATP) channels TM: Trabecular meshwork; SC: Schlemm’s canal

SC SC

SC TM

Neg con

TM

SUR2B

TM

Kir6.2

Possible active subunit combination: Kir6.2/SUR2B Roy Chowdhury U et al. IOVS 2011, 2013 (Page 3)

KATP channel: Cellular biosensors Sense cellular metabolic states  Increased cellular metabolism (closed)  Decreased cellular metabolism (open) Cellular functions  Contractile properties of smooth muscle  Formation of gap and tight junctions  Actin cytoskeleton rearrangement  Cellular endurance and recovery during stress (shear, oxidative, shock, pressure) (Page 4)

KATP channel openers  Cromakalim (CKL)  Diazoxide  Nicorandil

Vasodilator for acute/malignant hypertension, congenital hyperinsulinism, angina, etc.

 KATP channel opener diazoxide & nicorandil lowers IOP through the Kir6.2/SUR2B subunit containing KATP channels  Molecular structure of cromakalim is more conducive to chemical modifications that may make it more therapy friendly

Roy Chowdhury U et al. IOVS 2011, 2013 (Page 5)

Human anterior segment culture system Monitor pressure

Perfuse media

Pressure (mmHg)

40 35 30 25 20 15 10 5 0 0

1

2

3

4

5

6

7

8

Time (Days)

 Conventional outflow pathway  One-way fluid flow  Pressure dependent  Only human model (Page 6)

Pressure change (ex vivo) 30 Pressure (mmHg)

25

CKL

DMEM

20 15 10 5

Vehicle DMEM

0 CKL (2 µM) Vehicle

0

6

12

18

24

Time (1hr)

30

36

42

48

(Representative graph)

(Page 7)

CKL, CKLP1 lowers pressure (ex vivo)

CKL

CKLP1

35

35

n=11

Pressure (mmHg)

25

*

20 15 10 5

0h

24 h

0h

24 h

0

30 25

Pressure (mmHg)

30

n=5

*

20 15 10 5

0h

24 h

0h

24 h

0

Vehicle

CKL

Vehicle: 15.6 ± 4.9 to 15.6 ± 4.4 mmHg, (p=1.0) CKL: 19.3 ± 2.6 to 12.6 ± 3.0 mmHg, (p<0.001*)

Vehicle

CKL-P1

Vehicle: 21.8 ± 8.6 to 22.4 ± 9.6 mmHg, (p=0.37) CKL: 17.6 ± 6.0 to 8.0 ± 1.6 mmHg, (p<0.02*) (Page 8)

Optically active CKLP1 in mice (historical data from glaucoma therpeutics) 2 1 0

ΔIOP

-1 -2 -3 -4 CKL (n=5)

-5

CKL-P1 (R) (n=5) (+)CKL-P1 (n=10)

-6 1

2 Baseline

3

4

5

6

7

8

CKL (various forms)

9

10

11

12

13

Post treatment

Figure 2. Comparison of cromakalim and cromakalim prodrugs. Treatment of C57BL/6 mice with the optically active form of CKLP1 resulted in a pressure reduction that is similar to the parent compound cromakalim. The racemic form of CKLP1 also caused a pressure reduction however it was less than the optically active form of CKLP1 and cromakalim.

Whitlock et al. Journal of Ocular Pharmacology and Therapeutics, 25, 2009, 187.

(Page 9)

Effect of CKLP1 on IOP in Rabbits 20

% change compared to control eye

15 10 5 0 -5 -10 -15 -20 -25 -30

1

2

3

Pretreatment

4

5

6

7

(+)CKL-P1 (2.5 mM)

8

9

10

11

12

13

14

(+)CKL-P1 (5 mM)

50 µl bolus added as a single drop – once daily

15

16

17

Post treatment (Page 10)