USOO8865641B2 (12) United States Patent (10) Patent No.: US 8,865,641 B2 Pavlov et al. (45) Date of Patent: Oct. 21, 2014 (54) METHODS OF TREATMENT OF FATTY (2013.01); A61 K3I/46 (2013.01); A61 K LVER DISEASEBY PHARMACOLOGICAL 3 I/5513 (2013.01); A61K 31/167 (2013.01): ACTIVATION OF CHOLINERGIC PATHWAYS A6 IK3I/439 (2013.01) USPC ....... 514/1.1; 514/215: 514/220; 514/255.03; (75) Inventors: Valentin A. Pavlov, Bayside, NY (US); 514/278; 5.4394,51435,543 1 65 14/334: Kevin J. Tracey, Old Greenwich, CT 514471. 514/485; 514/614: 514/642 US) (58) Field of Classification Search ( None (73) Assignee: The Feinstein Institute for Medical See application file for complete search history. Research, Manhasset, NY (US) (56) References Cited (*) Notice: Subject to any disclaimer, the term of this U.S. PATENT DOCUMENTS patent is extended or adjusted under 35 U.S.C. 154(b) by 162 days. 6,610,713 B2 8/2003 Tracey 6,838,471 B2 1/2005 Tracey (21) Appl. No.: 13/523,519 7,238,715 B2 * 7/2007 Tracey et al. ................. 514,334 7,273,872 B2 9/2007 Tracey et al. 1-1. 7,662.965 B2 2/2010 Habgood et al. (22) Filed: Jun. 14, 2012 7,807,696 B2 10/2010 Al-Abed et al. O O 8,008.499 B2 8/2011 Habgood et al. (65) Prior Publication Data 2005/0125044 A1 6/2005 Tracey 2007/0173495 A1* 7, 2007 Palani et al. .................. 514,218 US 2012/0322719 A1 Dec. 20, 2012 2007/0213350 A1 9/2007 Tracey et al. 2009,0081314 A1 3, 2009 Wills 2011 OO15231 A1 1/2011 Al-Abed et al. Related U.S. ApplicationO O Data 2011/01661482011 0112128 A1 7/20115/2011 Tracey et al. (60) Provisional application No. 61/497,838, filed on Jun. 16, 2011. FOREIGN PATENT DOCUMENTS (51) Int. Cl. WO WO 2007OO1975 A1 : 1/2007 WO WO 2008/040548 A3 4/2008 A6 IK38/00 (2006.01) WO WO 2011006066 A1 * 1, 2011 AOIN 43/00 (2006.01) AOIN 43/46 (2006.01) OTHER PUBLICATIONS A6 IK3I/55 (2006.01) Guyenet S “Whole Helath Source: Choline and Fatty Liver” <http:// AOIN 43/62 (2006.01) wholehealthSource.blogspot.com/2010/11?choline-and-fatty-liver. A6 IK3I/4965 (2006.01) html> Accessed on the Internet Dec. 10, 2012. Published Nov. 29, AOIN 43/42 (2006.01) 2010.* A6 IK3I/44 (2006.01) Greenlee, W., et al., “Muscarinic agonists and antagonists in the AOIN 43/90 (2006.01) treatment of Alzheimer's disease.” Il Farmaco (56) pp: 247-250 AOIN 43/40 (2006.01) (2001). & 8 A6 IK3I/425 (2006.01) pay S.A. Slay liaisy's as AOIN 43/08 (2006.01) er, SV- state p Ca s 9-3 - A6 IK3I/34 (2006.01) Mice.” Mol Med 17(7-8):599-606 (Jul.-Aug. 2011). AOIN 47/10 (2006.01) * cited by examiner A6 IK3I/27 (2006.01) AOIN37/18 (2006.01) Primary Examiner — Maury Audet A6 IK3I/6 (2006.01) Assistant Examiner — Zachary J. Miknis AOIN33/12 (2006.01) (74) Attorney, Agent, or Firm — Hamilton, Brook, Smith & A6 IK3I/4 (2006.01) Reynolds, PC. A6 IK3I/444 (2006.01) A6 IK3I/495 (2006.01) (57) ABSTRACT A6 IK3I/34 (2006.01) A method of treating a fatty liver disease in a subject. The A6 IK3 L/4545 (2006.01) method comprises administering to the Subject an effective A6 IK3 L/46 (2006.01) amount of a cholinergic pathway stimulating agent, wherein A 6LX3/553 (2006.01) the fatty liver disease is selected from non-alcoholic fatty A6 IK3I/67 (2006.01) liver (NAFL), alcoholic fatty liver (AFL), non-alcoholic ste A6 IK3I/439 (2006.01) atohepatitis (NASH), alcoholic steatohepatitis (ASH), (52) U.S. Cl. NASH-associated liver fibrosis, ASH-associated liver fibro CPC ............... A6 IK3I/14 (2013.01); A61 K3I/444 sis, non-alcoholic cirrhosis, and alcoholic cirrhosis. (2013.01); A61 K3I/495 (2013.01); A61 K 3 I/55 (2013.01); A61 K3I/341 (2013.01); 29 Claims, 13 Drawing Sheets A6 IK3I/27 (2013.01); A61 K3I/4545 (2 of 13 Drawing Sheet(s) Filed in Color) U.S. Patent Oct. 21, 2014 Sheet 1 of 13 US 8,865,641 B2 FG. U.S. Patent Oct. 21, 2014 Sheet 2 of 13 US 8,865,641 B2 FG 2 U.S. Patent Oct. 21, 2014 Sheet 3 of 13 US 8,865,641 B2 (aamiasinouf) as euspoo : s 2 X { } {x : x is xi xi x) { {x x (f) fier Apog U.S. Patent US 8,865,641 B2 U.S. Patent Oct. 21, 2014 Sheet 6 of 13 US 8,865,641 B2 8 3. : 8 w : 3. X w r i & gx 3 & 8 : : « - - - - - - - - - - - - - - - . .----- - - - - - - - - - - 8;R &; : x 8 s: &s's & s: c U.S. Patent Oct. 21, 2014 Sheet 7 of 13 US 8,865,641 B2 c e (f) far dea xS xS. x S. : ex (if Yekaised U.S. Patent Oct. 21, 2014 Sheet 8 of 13 US 8,865,641 B2 i : U.S. Patent Oct. 21, 2014 Sheet 9 of 13 US 8,865,641 B2 U.S. Patent Oct. 21, 2014 Sheet 10 of 13 US 8,865,641 B2 ... c. it c it c is is Sun fielce) O Licenturiae pad ceded U.S. Patent Oct. 21, 2014 Sheet 11 of 13 US 8,865,641 B2 8 S. SSSSSSSSSSSSSS. SSkx: o: c grew r (fathasota is 8x8 pod K s: C e x-(6) ubiemK Apog U.S. Patent Oct. 21, 2014 Sheet 12 of 13 US 8,865,641 B2 s 3. 8: O eSEx. x. 388x Kei U.S. Patent Oct. 21, 2014 Sheet 13 of 13 US 8,865,641 B2 - 8: & i & ; : * & 8 : US 8,865,641 B2 1. 2 METHODS OF TREATMENT OF EATTY ergic pathway stimulating agent, wherein the fatty liver dis LIVER DISEASEBY PHARMACOLOGICAL ease is selected from non-alcoholic fatty liver (NAFL), alco ACTIVATION OF CHOLINERGC PATHWAYS holic fatty liver (AFL), non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), NASH-associated RELATED APPLICATION liver fibrosis, ASH-associated liver fibrosis, non-alcoholic cirrhosis and alcoholic cirrhosis. This application claims the benefit of U.S. Provisional Application No. 61/497,838, filed on Jun. 16, 2011. BRIEF DESCRIPTION OF THE DRAWINGS The entire teachings of the above application are incorpo rated herein by reference. 10 The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application GOVERNMENT SUPPORT publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. This invention was made with government Support under The foregoing will be apparent from the following more grant GM57226 awarded by the National Institutes of Health. 15 particular description of example embodiments of the inven The government has certain rights in the invention. tion, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout BACKGROUND OF THE INVENTION the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodi Fatty liverdisease is a condition characterized by excessive ments of the present invention. accumulation of lipids (fat) in liver. The build-up offat in liver FIG. 1 is a table listing the components of a low-fat diet. results in a range of clinical manifestations and progresses in FIG. 2 is a table listing the components of a high-fat diet. stages. Depending on etiology, each stage can be character FIG. 3A is a plot showing the body weight of low-fat diet ized as non-alcoholic or alcoholic. The progression begins fed (control mice) and treated with saline (LFD-S), mice on a with simple fatty liver, or Steatosis. This stage, generally 25 high-fat diet and treated with saline (HFD-S) and mice on a regarded as benign, is characterized by the increased appear high-fat diet and treated with galantamine (HFD-G) as a ance of fat in the liver. Fatty liver can be characterized as function of time. non-alcoholic (NAFL) or alcoholic (AFL). The next stage of FIG. 3B is a bar graph showing the food intake of the a fatty liver disease is a form of hepatitis known as Steato groups of mice under study before and after treatment. hepatitis, characterized by further fat accumulation and liver 30 FIG. 4 shows Table 1, which provides data showing effect tissue inflammation. Steatohepatitis can be non-alcoholic of galantamine on abdominal adiposity. (NASH) or alcoholic (ASH). Both NASH and ASH can lead FIG. 5A through 5F are bar plots showing the measured to the next stage of fatty liver disease, NASH-associated or levels of the indicated analytes in the bloodstream of mice ASH-associated fibrosis, respectively, which is characterized under study. by Scarring of the liver. Finally, fibrosis can progress to cir 35 FIG. 6A through 6D are bar plots showing the measured rhosis, which causes irreversible damage to the liver and is the levels of the indicated analytes in the bloodstream of mice most severe stage. Cirrhosis can be non-alcoholic or alco under study. holic. FIG. 7A and FIG. 7B are bar plots showing plasma ALT Common risk factors for fatty liver disease are obesity, level and liver weight, respectively, in mice under study. diabetes and drinking alcohol to excess. While the relation 40 FIG. 8 is a photograph showing liver gross appearance in ship between these factors is not fully understood, they can be the three groups of mice.