USOO9636359B2 (12) United States Patent (10) Patent No.: US 9,636,359 B2 Kenyon et al. (45) Date of Patent: May 2, 2017 (54) PHARMACEUTICAL COMPOSITION FOR (52) U.S. Cl. TREATING CANCER COMPRISING CPC ............ A61K 33/04 (2013.01); A61 K3I/095 TRYPSINOGEN AND/OR (2013.01); A61K 3L/21 (2013.01); A61K 38/47 CHYMOTRYPSINOGEN AND AN ACTIVE (2013.01); A61K 38/4826 (2013.01); A61 K AGENT SELECTED FROMA SELENUM 45/06 (2013.01) (58) Field of Classification Search COMPOUND, A VANILLOID COMPOUND None AND A CYTOPLASMC GLYCOLYSIS See application file for complete search history. REDUCTION AGENT (75) Inventors: Julian Norman Kenyon, Hampshire (56) References Cited (GB); Paul Rodney Clayton, Surrey U.S. PATENT DOCUMENTS (GB); David Tosh, Bath and North East Somerset (GB); Fernando Felguer, 4,514,388 A * 4, 1985 Psaledakis ................... 424/94.1 Glenside (AU); Ralf Brandt, Greenwith 4,978.332 A * 12/1990 Luck ...................... A61K 33,24 (AU) 514,930 (Continued) (73) Assignee: The University of Sydney, New South Wales (AU) FOREIGN PATENT DOCUMENTS (*) Notice: Subject to any disclaimer, the term of this KR 2007/0012040 1, 2007 patent is extended or adjusted under 35 WO WO 2009/061051 ck 5, 2009 U.S.C. 154(b) by 0 days. (21) Appl. No.: 13/502,917 OTHER PUBLICATIONS (22) PCT Fed: Oct. 22, 2010 Novak JF et al. Proenzyme Therapy of Cancer, Anticanc Res 25: 1157-1178, 2005).* (86) PCT No.: PCT/AU2O1 O/OO1403 (Continued) S 371 (c)(1), (2), (4) Date: Jun. 22, 2012 Primary Examiner — Erin M Bowers (74) Attorney, Agent, or Firm — Carol L. Francis; Khin (87) PCT Pub. No.: WO2O11?047434 K. Chin; Bozicevic, Field & Francis LLP PCT Pub. Date: Apr. 28, 2011 (57) ABSTRACT (65) Prior Publication Data The present invention generally relates to pharmaceutical US 2012/O251516 A1 Oct. 4, 2012 compositions containing a protease proenzyme and use thereof for treating cancer. The pharmaceutical composi (30) Foreign Application Priority Data tions are directed to compositions comprising a protease proenzyme and an active agent, the composition being Oct. 22, 2009 (AU) ................................ 2009905147 capable of providing a multi-functional approach for treating Jun. 17, 2010 (AU) ................................ 2O10902655 cancer. The pharmaceutical compositions are also directed to compositions comprising a first and a second protease (51) Int. Cl. proenzyme capable of activation at or near a Surface of a A6 IK33/04 (2006.01) tumor cell to enhance cell-to-cell adhesion of tumor cells, A 6LX 3L/095 (2006.01) effect proteolysis of tumor cells, or induce tumor cell (Continued) (Continued) ; : 2888 4.38 883: 88.388 8. 3888 23888 38.3388 4003 EK23S (e... stafa wa US 9,636,359 B2 Page 2 apoptosis, differentiation or immunorecognition, wherein Gura, Science 278(5340): 1041-1042 (1997).* the first protease proenzyme is chymotrypsinogen and the Novak et al., Anticancer Res. 25: 1157-1178 (2005).* second protease proenzyme is trypsinogen. The pharmaceu Extended European Search Report EP10824316.3, mailed Mar, 6. tical compositions are also directed to compositions com 2013 (11 pages). prising a first and second active agent each capable of International Preliminary Report on Patentability, mailed May 3, inducing intracellular activity in tumor cells. 2012 (8 pages). Novak, J.F. and Trnka, F. Proenzyme therapy of cancer. Anticancer 21 Claims, 47 Drawing Sheets Res. Mar.-Apr. 2005; 25(2A): 1157-77. European Patent Office Communication App No. 10 824 316.3- 1464, mailed Nov. 11, 2013 (7 pages). Beuth et al. (2001) “Impact of complementary oral enzyme appli cation on the postoperative treatment results of breast cancer (51) Int. Cl. patients—results of an epidemiological multicentre retrollective A6 IK 3L/21 (2006.01) cohort study” Cancer Chemother Pharmacol, 47:S45-S54. A6 IK 38/48 (2006.01) Cohen et al. (1999) “Oral Enzyme Therapy and Experimental Rat A6 IK 45/06 (2006.01) Mammary Tumor Metastasis' Life Sciences, 65(24):2603-1614. A6 IK 38/47 (2006.01) Gonzalez and Isaacs (1999) “Evaluation of Pancreatic Proteolytic Enzyme Treatment of Adenocarcinoma of the Pancreas. With Nutri (56) References Cited tion and Detoxification Support' Nutrition and Cancer, 33(2): 117 124. U.S. PATENT DOCUMENTS Gurkoff and McCabe (1974) “Preliminary study on the effects of combined hydrolytic enzyme agents on mouse Krebs-2 carcinoma 5,595,756 A * 1/1997 Bally et al. ................... 424/450 Journal AOA, 672-673. 5,858,357 A 1/1999 Trnka et al. Leipner and Saller (2000) "Systemic Enzyme Therapy in Oncology; 6,670,330 B1* 12/2003 Lampidis et al. .............. 514, 23 Effect and Mode of Action” Drugs, 59(4):769-780. 2004/00 18987 A1 1/2004 Hoffman et al. ............... 514f19 Popiela et al. (2001) “Influence of a complementary treatment with 2004/0167079 A1* 8, 2004 Tidmarsh ............... A61K 31.70 oral enzymes on patients with colorectal cancers—an epidemiologi 514, 23 cal retrollective cohort study” Cancer Chemother Pharmacol, 2005/0026852 A1 2/2005 Rustum ................ A61K 31,095 47:S55-S63. 514/34 Saruc et al. (2004) “Pancreatic Enzyme Extract Improves Survival 2007/0031398 A1* 2, 2007 Miller ..................... A23L 1,034 in Murine Pancreatic Cancer' Pancreas, 28:401-412. 424/94.63 Wald et al. (1998) “Proteinases Reduce Metastatic Dissemination and Increase Survival Time in C57BI6 Mice with the Lewis Lung OTHER PUBLICATIONS Carcinoma' Life Sciences, 63(17):237-243. Waldet al. (1998) “Polyenzyme preparation Wobe-Mugos(R inhibits Dreyer et al., J. Biol. Chem. 217: 527-540 (1955).* growth of solid tumors and development of experimental metastases Wald et al., Cancer Chemother. Pharmacol. 47(Suppl.): S16-S22 in mice' Life Sciences, 62(3):43-48. (2001).* Chabner et al., Nature Rev. 5: 65-72 (2005).* * cited by examiner U.S. Patent May 2, 2017 Sheet 1 of 47 US 9,636,359 B2 28 s}} 88: 80000 Figure A: CaCO2 cell standard curve {{}}. 2}}} Figure B : HEK293 cell. Standard curve U.S. Patent May 2, 2017 Sheet 2 of 47 US 9,636,359 B2 Figure C : OE33 ceili standard curve 3. : 3. Figure 1B: Panc1 cell standard curve U.S. Patent US 9,636,359 B2 Ei OD492; CJO CONCENTRAON FIGURE 3 U.S. Patent May 2, 2017 Sheet 4 of 47 US 9,636,359 B2 PANC B PANC EGTRE 4A U.S. Patent May 2, 2017 Sheet S of 47 US 9,636,359 B2 P&NC :: FEGERE AR U.S. Patent May 2, 2017 Sheet 6 of 47 US 9,636,359 B2 PANCT EGTJ RE 4 C U.S. Patent May 2, 2017 Sheet 7 of 47 US 9,636,359 B2 CACO REATMENT B 0.45 0.40 IC50: 2.52 R2. O.98 0.35 O.30 S 0.25 O20 O. 5 O. O. OO5 O 2 4. 6 CONCENTRATION Fig 5A: Treatment 8 U.S. Patent May 2, 2017 Sheet 8 of 47 US 9,636,359 B2 CACO TREATMENT J CSOE 2.38 R2: O.98 S O 2 4. 6 CONCENTRATION Fig 5B: ''reatment J U.S. Patent May 2, 2017 Sheet 9 of 47 US 9,636,359 B2 CACO REATMENT 0.55 0.50 0.45 IC 50: 2.48 R2 0.92 0.40 S 0.35 0.30 O25 0.15 O 2 4. 6 CONCENTRAION Fig 5C: Treatment. T U.S. Patent May 2, 2017 Sheet 10 of 47 US 9,636,359 B2 A C B FIGURE 6A A C B D FIGURE 6B U.S. Patent May 2, 2017 Sheet 11 of 47 US 9,636,359 B2 FIGURE 7 A FIGURE 7B U.S. Patent May 2, 2017 Sheet 12 of 47 US 9,636,359 B2 A. B FGUE 8A A C D B FIGURE 8B U.S. Patent May 2, 2017 Sheet 13 of 47 US 9,636,359 B2 FGUER 9A FIGURE 9B U.S. Patent May 2, 2017 Sheet 14 of 47 US 9,636,359 B2 FIGURE 1 OA FIGURE OB U.S. Patent May 2, 2017 Sheet 15 Of 47 US 9,636,359 B2 FIGURE A FIGURE 1B U.S. Patent May 2, 2017 Sheet 16 of 47 US 9,636,359 B2 A C D B FIGURE 12 U.S. Patent May 2, 2017 Sheet 17 Of 47 US 9,636,359 B2 FIGURE 13 U.S. Patent May 2, 2017 Sheet 18 of 47 US 9,636,359 B2 : : s w igiitiitii); if U.S. Patent May 2, 2017 Sheet 19 Of 47 US 9,636,359 B2 e 3. 8 x- sa. x: 're X. finitis. -tii" U.S. Patent May 2, 2017 Sheet 20 of 47 US 9,636,359 B2 re 38 s s:x. 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