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(12) United States Patent (10) Patent No.: US 8,940,687 B2 Naidu Et Al

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(12) United States Patent (10) Patent No.: US 8,940,687 B2 Naidu Et Al USOO8940687B2 (12) United States Patent (10) Patent No.: US 8,940,687 B2 Naidu et al. (45) Date of Patent: Jan. 27, 2015 (54) ANTIMICROBIAL ANGIOGENIN 5,286,487 A 2/1994 Vallee et al. COMPLEXES (ANGEX) AND USES THEREOF 7,074,7596,172,040 B2B1 7,1/2001 2006 Naidu 7,125,963 B2 10/2006 Naidu (71) Applicant: Naidu LP, Pomona, CA (US) 7,601,689 B2 10/2009 Naidu 7.956,031 B2 6, 2011 Naidu et al. (72) Inventors: A. Satyanarayan Naidu, Diamond Bar, 8,003,603 B2 8/2011 Naidu CA (US); A. G. Tezus Naidu, Santa 8,021,659 B2 9, 2011 Naidu et al. Ana, CA (US); A. G. Sreus Naidu, 8,309,080 B2 11/2012 Naidu et al. Santa Ana, CA (US 2007, 0141101 A1 6/2007 Nugent et al. anta Ana, CA (US) 2008/02540.18 A1 10, 2008 Naidu 2011/0200575 A1 8, 2011 Naidu et al. (73) Assignee: Naidu LP, Pomona, CA (US) 2011/0286986 A1 11/2011 Naidu et al. (*) Notice: Subject to any disclaimer, the term of this OTHER PUBLICATIONS patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. González-Chávez et al. ((International Journal of Antimicrobial 21) Appl. No.: 13/861,236 Agents 33 (2009) 301.e1-301.e8).* (21) ppl. No.: 9 Acharya, et al. “Crystal Structure of Human Angiogenin Reveals the Structural Basis for its Functional Divergence from Ribonuclease.” (22) Filed: Apr. 11, 2013 Proc. Natl. Acad. Sci. USA, vol. 91, pp. 2915-2919, Apr. 1994. O O Hu, et al. A Putative Angiogenin Receptor in Angiogenin-Responsive (65) Prior Publication Data Human Endothelial Cells, Proc. Natl. Acad. Sci. USA. vol. 94, pp. US 2013/0287754 A1 Oct. 31, 2013 2204-2209, Mar. 1997. King, et al. Neovascularisation of the Meniscus with Angiogenin, An Related U.S. Application Data Experimental Study in Rabbits, The Journal of Bone and Joint Sur (60) Continuation of application No. 13/185.115, filed on gery, vol. 7-B, No. 4, pp. 587-590, Jul. 1991. -- Jul. 18, 2011, now Pat. No. 8,440,183, which- is0 a Maiti, et al. “Isolation ands Characterization of an Angiogenin-Like division- - - of application No. 12/554.602, filed on Sep. 4, pp.Protein 283-288, from Goat2002 Plasma.” Protein and Peptide Letters, vol.9, No. 4. 2009, now Pat. No. 8,003,603, which is a division of - .N. lication No. 11/734,729, filed Apr. 12, 2007 Moroianu, et al. "Nuclear Translocation of Angiogenin in Proliferat app ly g 6O168 9. , Illed on Apr. 12, s ing Endothelial Cells is Essential to its Angiogenic Activity.” Proc. now Pat. No. 7,601,689. Natl. Acad. Sci. USA, vol. 91, pp. 1677-1681, Mar. 1994. (51) Int. Cl R.Nagarajan, Langmuir 2000, 16, 6400-6415. A6 IK38/54 (2006.01) Sgt. The Angiogenins.” Cell. Mol. Life Sci., vol. 54, pp. 811 C07K I4/55 (2006.01) Soncin,. et. v. al. “Interaction of Heparin with Human Angiogenin.” The AA6 6LX39/395 IK38/43 (2006.01) J y 1 of Biologicalogical Chemistry,Chemi vol.1. 272, No. 15,15 pp. 98.18-9824,98.18-9824 GOIN33/68 (2006.01) pr. 1, (52) is 40 (2006.01) * cited by examiner CPC ............... A61K 38/54 (2013.01); C07K 14/515 (2013.01); A61K.39/3955 (2013.01); G0IN Primary Examiner — Bridget E Bunner 33/6854 (2013.01) Assistant Examiner — Fozia Hamud USPC ............. 514/2.3; 424/94.3: 514/2.4: 514/2.5; 51.4/26: 514/2.7: 514/28: 514/34 (74) Attorney, Agent, or Firm — Knobbe, Martens, Olson & • Ys • s • Y-s Bear LLP (58) Field of Classification Search Ca None See application file for complete search history. (57) ABSTRACT (56) References Cited Antimicrobial compositions based upon stabilized angioge nin compositions also contain osteopontin and antimicrobial U.S. PATENT DOCUMENTS proteins such as lactoperoxidase (LPO), myeloperoxidase 4,900,673 A 2/1990 Harper et al. (MPO), salivary peroxidase (SPO) and lysozyme. 4,966,964 A 10/1990 Shapiro et al. 5,171,845. A 12/1992 Spiket al. 5,270,204 A 12/1993 Vallee et al. 13 Claims, 5 Drawing Sheets U.S. Patent Jan. 27, 2015 Sheet 1 of 5 US 8,940,687 B2 FIGURE-1: Isolation and purification of ANG 0.20 100 0.15 80 S 60 G O O. 10 8. N Q 40 ?t 3 0.05 SS CC U 20 0.00 O -0.05 20 30 40 50 60 Time (min) U.S. Patent Jan. 27, 2015 Sheet 2 of 5 US 8,940,687 B2 FIGURE-2: Biotinylated ANG-LF Interactions 0.0006 0.0004 0.0002 0.0000 0.0009 0.0006 0.0003 biotin-ANG interaction with LF 0.0000 O 10 20 30 40 50 60 70 Biotinylated-Protein (M) U.S. Patent Jan. 27, 2015 Sheet 3 of 5 US 8,940,687 B2 FIGURE-3: ANGex Detection by Turbidometry 0.100 F-ANG Interaction 0.095 0.090 Eaw s 1 c N Xa s 0. s 0.085 h o O. 8 c 7 1 2 3. 4. s ANGELF molar ratio 0.080 O 1 2 3 4 5 ANG/(LF) molar ratio U.S. Patent Jan. 27, 2015 Sheet 4 of 5 US 8,940,687 B2 FIGURE-4: Detection of ANGex formation 0.15 O. 10 0.05 O.00 -0.05 60 65 70 75 80 85 90 95 100 Time (min) U.S. Patent Jan. 27, 2015 Sheet 5 of 5 US 8,940,687 B2 FIGURE-5: Antioxidant activity of ANGex Time (h) O ANG r = 0.6533 O ANGex (with 25aM of LF) r = 0.6668 V ANGex (with 75M of LF) r = 0.2986 ANGex (with 125M of LF) r = 0.3855 O.O 0.2 O4 O.6 Angiogenin mM US 8,940,687 B2 1. 2 ANTIMICROBAL ANGOGENIN about 0.3 ug/mL with a fast turnover rate and a half-life <5 COMPLEXES (ANGEX) AND USES THEREOF min. ANG can induce most of the events necessary for the formation of new blood vessels. It binds avidly to endothelial RELATED APPLICATIONS cells and stimulates cell migration and invasion. ANG pro motes cell proliferation and differentiation; mediates cell This application is a continuation of U.S. application Ser. adhesion and activates cell associated proteases; and also No. 13/185,115, filed Jul.18, 2011, now U.S. Pat. No. 8,440, induces plasminogen activator and thereby, the plasmin sys 183, issued May 14, 2013, which is a divisional of U.S. tem promoting migration and tubular morphogenesis of application Ser. No. 12/554,602, filed Sep. 4, 2009, now U.S. endothelial cells. Exogenous ANG is transported into the Pat. No. 8,003,603, issued Aug. 23, 2011, which is a divi 10 nucleus of endothelial cells. The nuclear translocation results sional of U.S. application Ser. No. 1 1/734,729, filed Apr. 12, in accumulation of the ANG in the nucleolus. Transportation 2007, now U.S. Pat. No. 7,601,689 issued Oct. 13, 2009. All of the above listed applications are incorporated herein by of ANG from the cell surface into the nucleus and subse reference in their entirety. quently to the nucleolus is critical for its angiogenic activity. 15 The import of ANG from the cytosol to the nucleus is signal BACKGROUND OF THE INVENTION dependent, carrier mediated and energy-dependent, active transport process (Hu GF, et al., Proc. Natl. Acad. Sci. USA 1. Field of the Invention 94:2204-2209, 1997: Moroianu J, et al., Proc. Natl. Acad. Sci. The invention relates to protein stabilization, particularly USA 91: 1677-1681, 1994). stabilization of angiogenin by immobilization on natural Sub ANG is a potent inducer of neo-vascularization and the strates which includes but not limited to proteins, polysac only angiogenic molecule known to exhibit ribonucleolytic charides, lipids and polyphenols. activity. Its overall structure, as determined at 2.4A, is similar 2. Description of the Related Art to that of pancreatic RNase A, but it differs markedly in Angiogenesis and vasculogenesis are two primary path several distinct areas, particularly the ribonucleolytic active ways in the development and maintenance of mammalian 25 center and the putative receptor binding site, both of which health. The angiogenic role is to Supply and Support tissue are critically involved in biological function. Most strikingly, with ample vasculature, thus providing a route of access for the site that is spatially analogous to that for pyrimidine the transportation of essential nutrients, including oxygen binding in RNase A differs significantly in conformation and and the removal of metabolic waste in a Sustained manner. is "obstructed by Gln-117. Movement of this and adjacent Angiogenesis is a strictly regulated, multi-step process that 30 residues may be required for substrate binding to ANG and, occurs during normal physiology Such as wound healing, hence, constitute a key part of its mechanism of action pregnancy, and development. (Acharya K R, et al., Proc. Natl. Acad. Sci. USA 91:2915 Angiogenin (ANG) has been shown to be a key mediating 2919, 1994; Russo N, et al., Proc. Natl. Acad. Sci. USA factor in the underlying cascade of chemical events leading to 91:2920-2924, 1994). angiogenesis, which makes it a very important precursor mol 35 X-ray diffraction and mutagenesis results have shown that ecule for both muscle development and vascular generation. the active site of the human protein is obstructed by Gln-117 ANG is a 14-kDa, basic heparin-binding protein and a mem and imply that the C-terminal region of ANG must undergo a ber of the pancreatic ribonuclease (RNase) superfamily.
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