US 2010.0075894A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0075894 A1 Hotamisligil et al. (43) Pub. Date: Mar. 25, 2010 (54) REDUCINGER STRESS IN THE TREATMENT A63L/92 (2006.01) OF OBESITY AND DABETES A 6LX 3/575 (2006.01) A63/04 (2006.01) (75) Inventors: Gökhan S. Hotamisligil, Wellesley, A6II 3/55 (2006.01) MA (US); Umut Ozcan, Brookline, A613/60 (2006.01) MA (US) A6IP3/10 (2006.01) A6IP3/04 (2006.01) Correspondence Address: A6IP 9/10 (2006.01) EDWARDSANGELL PALMER & DODGE LLP (s2 usic... 514/4; 435/7.2:436/86; 514/570; P.O. BOX SS874 514/169; 514/740: 514/171; 514/635: 514/165 BOSTON, MA 02205 (US) (57) ABSTRACT (73) Assignee: HARYARPN.YERSITY, Endoplasmic reticulum stress has been found to be associated ambridge, (US) with obesity. Therefore, agents that reduce or prevent ER (21) Appl. No.: 12/541,020 stress may be used to treat diseases associated with obesity including peripheral insulin resistance, hypergylcemia, and (22) Filed: Aug. 13, 2009 type 2 diabetes. Two compounds which have been shown to reduce ER stress and to reduce blood glucose levels include Related U.S. Application Data 4-phenyl butyric acid (PBA), tauroursodeoxycholic acid (TUDCA), and trimethylamine N-oxide (TMAO). Other (63) Continuation of application No. 1 1/227.497, filed on compounds useful in reducing ER stress are chemical chap Sep. 15, 2005, now abandoned. erones Such as trimethylamine N-oxide and glycerol. The (60) Provisional application No. 60/610,093, filed on Sep. E. invention provides methods of treating a subject suf 15, 2004. ering from obesity, hyperglycemia, type 2 diabetes, or insu s lin resistance using ER stress reducers such as PBA, TUDCA, O O and TMAO. Methods of screening for ER stress reducers by Publication Classification identifying agents that reduce levels of ER stress markers in (51) Int. Cl. ER stressed cells are also provided. These agents may find use A6 IK 38/28 (2006.01) in methods and pharmaceutical compositions for treating GOIN 33/53 (2006.01) obesity-associated diseases. LEAN Ob/Ob RD HFD 3. sik--XBP-1(S) GRP78 28S 18S LEAN Ob/Ob IGRP78 28S --Tot-JNK 18S Patent Application Publication Mar. 25, 2010 Sheet 1 of 22 US 2010/0075894 A1 qO/GO qO/qONWET Patent Application Publication Mar. 25, 2010 Sheet 2 of 22 US 2010/0075894 A1 IP: IRS-1 E. E.s R.ss IB:IP: IRS-1IRS-1 ser307 IB: IRS-1 ser307 AIP: IRS-1 IP: IRS-1 EgySESSIB: IRS-1 EIB. IRS-1 IP: pY IB: IRS-1 IP: pY IB: pY Patent Application Publication Mar. 25, 2010 Sheet 3 of 22 US 2010/0075894 A1 is is 2,: ESSEEE i E.SR g Tot-JNK O 1 h FIG. 3A Tun: IP: IRS-1 s IB: p-IRS-1Ser307 IP: IRS-1 *." IB: IRS-1 IB: p-IRS-1 ser307 IP: IRS-1 IB: IRS-1 Patent Application Publication Mar. 25, 2010 Sheet 4 of 22 US 2010/0075894 A1 IRE-1Ot/ IRE-1O/ Ins: - - - - - - - - - - - - - - IP: IRS-1 IB: pY IP: IRS B: IRS-1 N - DIRE-1 +/- 5g.DN OIRE-1 -/- O.8 25U. 0.4 O.O Patent Application Publication Mar. 25, 2010 Sheet 5 of 22 US 2010/0075894 A1 3OO U) g 250 c 3 S. S 2 15O O Ye - O 1OO nS 5O O X PBS 4-PBA FIG. 4A 350 300 g a 250 - O3 N.G 200 t5 a1E 150 g 1OO O O 15 3O 6O 90 12O Minutes After Insulin Injection FIG. 4B Patent Application Publication Mar. 25, 2010 Sheet 6 of 22 US 2010/0075894 A1 Body Weight During Trial ex - - - - - - - - - - - - - - - - - - - V - - - ----------------------a ma ------mava --- m V-w 500 OO S. 50.00 ... -- ob/ob PBS si cleanPBS Eg 40.00-TV. to -A--- ob/oblean PBS PBA 3350- 300 His ean PBA g 30.00 * -e- lean PBA S 58 & 20.00 ---------r 3 ra 15 & 10.00 as 19 O.OO 8 R O Day 0 Day 20 S S R Days A FIG. 5B so. ITT at 15th day. Insulin Levels i 300 14 s -s 12 ob/ob PBS is 250 510 Zob/ob PBA 200- -- ob/ob pbs S. Olean PBS Ne -- ob/ob pba S 8 Sean PBA S; 150 -A-lean pbs 6 8 100 r-e-lean pba 4 5 50 - 2 8 O O i O 30 60 90 120 Minutes After Insulin Injection FIG. 5D Patent Application Publication Mar. 25, 2010 Sheet 7 of 22 US 2010/0075894 A1 Blood Glucose Levels ob/ob(PBS) 4 O O ob/ob(TUD) O Lean (PBS) Lean (TUD) 2 O O 54O ckk ck k ck >k 450 KK ck k ckk -- ob/ob (TUDCA) 36O -H ob/ob (PBS) -A- Lean (PBS) 27O -X - Lean (TUDCA) 18O 9 O O O 15 3O 60 90 120 Minutes After Insulin Injection FIG. 6B Patent Application Publication Mar. 25, 2010 Sheet 8 of 22 US 2010/0075894 A1 5OO 400 3OO 2OO 1OO Days of treatment of diabetic mice with TMAO FIG. 7A 16. O PBS OTMAO S. 12.. O - s 8. O O39 Na Days of treatment of diabetic mice with TMAO FIG. 7B Patent Application Publication Mar. 25, 2010 Sheet 9 of 22 US 2010/0075894 A1 6 O O 3O O 200 1 OO O PBS PBA (200mg) MET (200mg) PBA--MET (200) Treatment (diabetic mice, ob/ob, treated for 11 days at 200 mg/kg/day dose) Glu (mM) STZ. al -- O 5 10 25 75 var-Y mall GRP78 sausales as 28s is 18S Patent Application Publication Mar. 25, 2010 Sheet 10 of 22 US 2010/0075894 A1 FIG. 1 OA Lean Ob/Ob Patent Application Publication Mar. 25, 2010 Sheet 11 of 22 US 2010/0075894 A1 IB: pY : IB: IRS-1Pser a , IP: IRS-1 s IB : IRS-1 Control Insulin s O Ins+Tun O 1OO 200 Phospho/Total ?yS 200 k IP: IRS-1 IB: IRS-1Pser 'g. 120 s IP: IRS-1 th 40 IB: IRS-1 Thap: - - + + JNKi: - - - - IP: pY IB: IRS-1 .. IP: pY IB: pY Patent Application Publication Mar. 25, 2010 Sheet 12 of 22 US 2010/0075894 A1 TUn: - -- -- -- Time(hr): O 1. 2 4 p-c-Jun - IP: IRS-1 : IB: IRS-1 pSer307 IP: IRS-1 IB: IRS-1 p-c-Jun s IP: IRS-1 IB: IRS-1 pSer307 IP: IRS-1 IB: IRS-1 IRE-1Ot/ IRE-O/ Tun; O O 1 2 3 4 O O 1 2 3 4 (hr) Ins: - - - - IP: IRS-1 IB; pY IP: IRS-1 IB: IRS-1 5OO IRE1 -/- k 4OO IRE-1 - A.A -- 3OO i 2OO 1OO Tun: O O 1. 2 3 4 (hr) Ins: - H -- -- -- -- FIG. 11H Patent Application Publication Mar. 25, 2010 Sheet 13 of 22 US 2010/0075894 A1 : IRS-1 : pY : IRS-1 : IRS-1 pSer307 : IRS-1 : IRS-1 IR : pY : IR : IR IP: IRS-1 IB: IRS-1 pSer307 IP: IRS-1 IB : IRS-1 FIG. 12B Patent Application Publication Mar. 25, 2010 Sheet 14 of 22 US 2010/0075894 A1 - FDOX- -DOX Tun(2g/ml): - + + + + + - + + + + + Time(hr): O 1 2 3 4 5 O 1 2 3 4 5 FIG. 13A --DOX -DOx Tun (2ug/ml): - + + + + + + - + + + + + + Time(hr): O 0.51 2 3 4 5 O 0.51 2 3 4 5 : - - JNK FIG. 13C XBP-1/ XBP-t/- Tun(O.5ug/ml): - + + + + + + - + + + + + + Time(hr): O O.5 1 2 3 4 5 O 0.51 2 3 4 5 FIG. 13D Patent Application Publication Mar. 25, 2010 Sheet 15 of 22 US 2010/0075894 A1 --DOX -DOX Tun (2g/ml): - + + - - - -- Time(hr): O 4 5 O 4 5 S 1000 D-Dox Y pSer307 '3. 600 ", 400 U?) St IRS-1 a 200 FIG 13E Time(hr): O +Dox 500 400 Uh 300 9, 200 1OO i Time(hr): 5 Tun: - -H Ins: - H XBP-1-/- XBP-1t/t EXBP-1 -/- N Tun (O.5ug/ml): - + + - + + O 2500 XBP-1 /+ Time(hr): O 4 5 O 4 5 8 2000 w IRS-1 S. 15OO H OOO U : g 5OO Time(hr): O 4 5 Tun: - -- -- Ins: - -- -- FIG. 13H Patent Application Publication Mar. 25, 2010 Sheet 16 of 22 US 2010/0075894 A1 --Dox -DOX Tun (2g/ml): O O 0.5 1 2 3 4 O O 0.5 1 2 3 4 (hr) InS: - - - - - - - - - - - - - -- Ogg Qup XBP-1/- XBP-1+/+ Tun (0.5ug/ml): O O O.5. 1 2 3 4 O O 0.5 1 2 3 4 (hr) Ins: - - - - - - - - - - - - - - s AAas? as, ? IP:IB: pyIR IP: IR IB: IR FIG. 14B Patent Application Publication Mar. 25, 2010 Sheet 17 of 22 US 2010/0075894 A1 3 sks: 18O s kick S 2 kk 1. 2 O S. k 1 . 6 O O 6 O O O O O 4 8 1216 20 O 4 8. 21620 O 4 8 121620 O 4 8 12 1620 Weeks on Diet Weeks on Diet Weeks on Diet Weeks on Diet FIG. 15A FIG. 15C FIG. 15D 540 540 240 240 .." ck/X sk sk kk 5 3 6 O Yag360 k 1. 6 O sk o 8 O 8 18O 8 O 8 O P ( O O O O O 1530. 6090120 O 1530 6090120 O 1530 60 90120 O 1530 60 90120 Time After Glucose (min) Time After Glucose (min) Time After Insulin (min) Time After Insulin (min) FIG. 15E FIG. 15F FIG. 15G FIG. 15H Patent Application Publication Mar.