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(12) United States Patent (10) Patent No.: US 9,474,773 B2 Neu Et Al

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(12) United States Patent (10) Patent No.: US 9,474,773 B2 Neu Et Al US009474773B2 (12) United States Patent (10) Patent No.: US 9,474,773 B2 Neu et al. (45) Date of Patent: Oct. 25, 2016 (54) LACTOBACILLUS SUPPLEMENT FOR (52) U.S. Cl. ALLEVIATING TYPE 1 DABETES CPC ........... A61K 35/747 (2013.01); A23C 9/1234 (2013.01); A23C 19/0323 (2013.01); A23G (75) Inventors: Josef Neu, Gainesville, FL (US); 9/363 (2013.01); A23L I/3014 (2013.01); Graciela Liliana Lorca, Gainesville, CI2N 1/20 (2013.01); A23 V 2002/00 FL (US); Eric W. Triplett, Gainesville, (2013.01); FL (US); Mark A. Atkinson, (Continued) Gainesville, FL (US). Desmond A. (58) Field of Classification Search Schatz, Gainesville, FL (US) None See application file for complete search history. (73) Assignee: UNIVERSITY OF FLORIDA RESEARCH FOUNDATION, INC., (56) References Cited Gainesville, FL (US) U.S. PATENT DOCUMENTS (*) Notice: Subject to any disclaimer, the term of this 5,200,318 A * 4/1993 Rabin .................. GON 33.564 patent is extended or adjusted under 35 435/4 U.S.C. 154(b) by 8 days. 5,556,785 A 9/1996 Kishida (21) Appl. No.: 13/144,028 (Continued) FOREIGN PATENT DOCUMENTS (22) PCT Filed: Feb. 18, 2010 WO WO 2004O76615 A2 * 9, 2004 (86). PCT No.: PCT/US2010/0245.75 WO WO 2008, 141989 11/2008 S 371 (c)(1), (2), (4) Date: Oct. 4, 2011 OTHER PUBLICATIONS Takeshi Matsuzaki, Yuriko Nagata, Shouichi Kado, Kazumi Uchida. (87) PCT Pub. No.: WO2010/096550 Ikuo Kato, Shusuke Hashimoto and Teruo Yokokura, Prevention of PCT Pub. Date: Aug. 26, 2010 onset in an insulin-dependent diabetes mellitus model, NOD mice, by oral feeding of Lactobacillus casei, 1997, Acta Pathologica, (65) Prior Publication Data Microbiologica et Immunological Scandinavica, vol. 105, pp. 643 649* US 2012/O183513 A1 Jul. 19, 2012 (Continued) Related U.S. Application Data Primary Examiner — Ralph Gitomer (60) Provisional application No. 61/153,516, filed on Feb. Assistant Examiner — Trent Clarke 18, 2009, provisional application No. 61/297,480, (74) Attorney, Agent, or Firm — Saliwanchik, Lloyd & filed on Jan. 22, 2010. Eisenschenk (57) ABSTRACT (51) Int. Cl. Isolated Lactobacillus strains are useful in preventing or A6 IK 35/74 (2015.01) delaying the development of Type 1 Diabetes (T1D). A CI2N L/20 (2006.01) probiotic composition comprising the Lactobacillus strains A6IP3/10 (2006.01) and use of the composition in T1D prevention are provided. (Continued) 13 Claims, 12 Drawing Sheets rero or - r n - 60 40 : in vie: is is is f Age (weeks) US 9,474,773 B2 Page 2 (51) Int. Cl. Autoimmune Diabetes Mellitus: Caveat Emptor and Primum non A 6LX 35/747 (2015.01) Nocere, 2001, Clinical Immunology, vol. 100, No. 2, pp. 134-143.* A23C 9/23 (2006.01) Brugman et al., “Antibiotic treatment partially protects against type A23C 9/032 (2006.01) 1 siabetes in the Bio-Breeding diabetes-prone rat. Is the gut flora A23G 9/36 (2006.01) involved in the development of type 1 diabetes?”. Diabetologia, A2.3L I/30 (2006.01) 2006, vol. 49, pp. 2105-2108. Calcinaro et al., “Oral probiotic administration induces interleukin A61 K 35/00 (2006.01) 10 production and prevents spontaneous autoimmune diabetes in the (52) U.S. Cl. non-obese diabetic mouse”. Diabetologia, 2005, vol. 48, pp. 1565 CPC ......... A23Y2220/43 (2013.01); A23Y 2220/71 1575. (2013.01); A61 K 2035/115 (2013.01) Like et al., “Influence of environmental viral agents on frequency and tempo of diabetes mellitus in BB/Wor rats', Diabetes, 1991, (56) References Cited vol. 40, No. 2, pp. 259-262, abstract. U.S. PATENT DOCUMENTS Matsuzaki et al., “Prevention of onset in an insulin-dependent diabetes mellitus model, NOD mice, by oral feeding of Lactobacil 6.214.336 B1 4/2001 Bukowska et al. lus casei'. Acta Pathologica, Microbiologica et Immunologica 6,808,703 B2 10/2004 Park et al. Scandinavica (APMIS), 1997, vol. 105, pp. 643-649. 7,001,756 B1 2/2006 Hsu et al. McInerney et al., “Prevention of insulitis and diabetes onset by 7,189,390 B2 3/2007 Zink et al. treatment with complete Freund's adjuvant in NOD mice', Diabe 7,390,519 B2 6, 2008 Collins et al. 2003/0235559 A1* 12, 2003 Sobol et al. ................. 424,934 tes, 1991, vol. 40, No. 6, pp. 715-725, abstract. 2005, 0100531 A1 5, 2005 Bienenstock Sadelain et al., “Prevention of type I diabetes in NOD mice by 2006/0270020 A1 11/2006 Boileau et al. adjuvan immunotherapy'. Diabetes, 1990, vol. 39, No. 5, pp. 2008.0193485 A1 8, 2008 Gorbach et al. ........... 424,234.1 583-589, abstract. Sadelain et al., “Prevention of Diabetes in the BB Rat by Early Immunotherapy Using Freund's Adjuvant”. Journal of Autoimmu OTHER PUBLICATIONS nity, 1990, vol. 3, pp. 671-680. NPL pdf document “Guidance memorandum Mar. 4, 2014” Salminen et al., “Intestinal colonistation, microbiota and future accessed Mar. 27, 2014 from http://www.uspto.gov/patents/law? probiotics?”. Asia Pacific Journal of Clinical Nutirtion, 2006, vol. exam/myriad-mayo guidance.pdf. 15, No. 4, pp. 558-562. JP 4044975 B, issued Feb. 6, 2008, NPL pdf document has JPO Schwartz et al., “Comment on: Brugman S. et al. (2006) Antibiotic English translation attached.* treatment partially protects against type 1 Siabetes in the Bio Jo Satoh, Shigeki Shintani, Keiichiro Oya, Shun-Ichi Tanaka, Breeding diabetes-prone rat. Is the gut flora involved in the devel Toshima Nobunaga, Takayoshi Toyota, and Yoshio Goto, Treatment opment of type 1 diabetes? Diabetologia, 49.2105-2108”. With Streptococcal Preparation (OK-432) Suppresses Anti-Islet Diabetologia, 2007, vol. 50, pp. 220-221. Autoimmunity and Prevents Diabetes in BB Rats, Diabetes, vol. 37. Toshihiko et al., Effects of the probiotic strain Lactobacillus Sep. 1988, pp. 1188-1194.* johnsonii Strain Lal on autonomic nerves and blood glucose in rats, Amal B. Shori, Antioxidant activity and viability of lactic acid Life Sciences, 2006, vol. 79, pp. 1963-1967. bacteria in Soybean-yogurt made from cow and camel milk, Journal Wicker et al., “Genetic Control of Diabetes and Insulitis in the of Taibah University for Science 7 (2013) 202-208.* Nonobese Diabetic (NOD) Mouse'. The Journal of Experimental “The Nutritional Composition of Dairy products” by The Dairy Medicine, 1987, vol. 165, pp. 1639-1654. Council, published online Aug. 27, 2003 according to Google.* Yadav et al., “Antidiabetic effect of probiotic dahi containing Kin Kwan Lai, Graciela L. Lorca, and Claudio F. Gonzalez, Lactobacillus acidophilus and Lactobacillus casei in high fructose Biochemical Properties of Two Cinnamoyl Esterases Purified from fed rats'. Nutrition, 2007, vol. 23, pp. 62-68. a Lactobacillus johnsonii Strain Isolated from Stool Samples of Vaarala et al., “The “Perfect Storm” for Type 1 Diabetes: The Diabetes-Resistant Rats, 2009, Applied and Environmental Micro Complex Interplay Between Intestinal Microbiota, Gut Permeabil biology, vol. 75, No. 15, pp. 5018-5024, published online Jun. 5, ity, and Mucosal Immunity', 2008, Diabetes, vol. 57, No. 10, pp. 2009. 2555-2562. Dale L. Greiner, Aldo A. Rossini, and John P. Mordes, Translating Data from Animal Models into Methods for Preventing Human * cited by examiner U.S. Patent Oct. 25, 2016 Sheet 1 of 12 US 9,474,773 B2 r w –[][][ ----UG U.S. Patent Oct. 25, 2016 Sheet 2 of 12 US 9,474,773 B2 FG 2A-1 PCA - Qualitative - A 0.10 - OOC -010 -0.2) FIG. 2A-2 PCA - Quantitative - A s {} BioBreeding Diabetes prone O BioBreeding Diabetes resistant U.S. Patent Oct. 25, 2016 Sheet 3 of 12 US 9,474,773 B2 FG. 2B-1 PCA - Guaitative - B 0. 2 * s c :: ** is 0,10 t E :- * 0.00" al x isse - - - - - o - . O ss . * - ....s. ------- is -0.20 s (n.30" 0.40 FIG. 2B-2 PCA Quantiative - B g - C.. BioBreeding Diabetes prone O BioBreeding Diabetes resistant U.S. Patent Oct. 25, 2016 Sheet 4 of 12 US 9,474,773 B2 SSeu3 w so L r o te t w re y o - - - o s se fill f. ?h C f f C wasI 'r'. r fl. 1. C. C. r - - S. val ra ir y c c s Xepu ASJewp Jewee w uouuleuS U.S. Patent Oct. 25, 2016 Sheet S of 12 US 9,474,773 B2 FIG. 4A 5 3.4 ww 2 Lactohacilius spp. Bifidobacteri in spp. BB-F BB-OR Lactobacilius spp. Bifidobacterium spp. U.S. Patent US 9,474,773 B2 3e3OeOOOOOuluny U.S. Patent Oct. 25, 2016 Sheet 7 of 12 US 9,474,773 B2 F.G. 6 1OO 468OOO 2 O Control L. johnsonii L. johnsonii L. reuteri L. reuteri 106 108 106 108 FIG. 7 OnSet Of T 1D Post-weaning (W) Pre-weaning 2 22 69 110 141 U.S. Patent Oct. 25, 2016 Sheet 8 of 12 US 9,474,773 B2 FIG. 8A 00 O Age (weeks) FIG. 8B in was a in an in a 20 is Age (weeks) U.S. Patent Oct. 25, 2016 Sheet 9 of 12 US 9,474,773 B2 FG. 9A (eu snffizºqoqooT andeq?enon?oaºequaquad} Healthy Diabetic FG. 9B Healthy Diabetic U.S. Patent Oct. 25, 2016 Sheet 10 of 12 US 9,474,773 B2 FG 1 OA E. : SO Argth it Depth FIG 1 OB sessesseggererees E. Jahrsarif Healthy Control g F.G. 10C l . Cai cilii U.S. Patent Oct. 25, 2016 Sheet 11 of 12 US 9,474,773 B2 G : l \ sessessessessessessessessessereasteresarestoressessessessee s: reer & NOS COX2 Sod1 Sod2 Gpx1 Cat GR FIG 11B Healthy L. johnsonii Control Diabetic iNOS B-actin U.S. Patent Oct. 25, 2016 Sheet 12 of 12 US 9,474,773 B2 FIG.
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