WO 2014/134225 A2 4 September 2014 (04.09.2014) P O P CT
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(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2014/134225 A2 4 September 2014 (04.09.2014) P O P CT (51) International Patent Classification: (74) Agents: KABLER, Kevin et al; Fenwick & West LLP, A61K 9/00 (2006.01) Silicon Valley Center, 801 California Street, Mountain View, CA 94041 (US). (21) International Application Number: PCT/US20 14/0 18807 (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (22) International Filing Date: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, 26 February 2014 (26.02.2014) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, (25) Filing Language: English DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, (26) Publication Language: English KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, (30) Priority Data: MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, 61/769,429 26 February 2013 (26.02.2013) OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, 61/769,444 26 February 2013 (26.02.2013) SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, 61/808,650 5 April 2013 (05.04.2013) TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (71) Applicant: PRONUTRIA, INC. [US/US]; 840 Memorial Drive, Third Floor, Cambridge, MA 02 139 (US). (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (72) Inventors: SILVER, Nathaniel; Pronutria, Inc., 840 M e GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, morial Drive, Third Floor, Cambridge, MA 021 39 (US). UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, HAMILL, Michael; Pronutria, Inc., 840 Memorial Drive, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, Third Floor, Cambridge, MA 02139 (US). SAMAYOA, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, ΓΓ, LT, LU, LV, Philip; Pronutria, Inc., 840 Memorial Drive, Third Floor, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, Cambridge, MA 02139 (US). HOU, Jay; Pronutria, Inc., TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, 840 Memorial Drive, Third Floor, Cambridge, MA 02139 KM, ML, MR, NE, SN, TD, TG). (US). HAMM, Luke; Pronutria, Inc., 840 Memorial Drive, Published: Third Floor, Cambridge, MA 02139 (US). BERRY, Dav¬ id; Pronutria, Inc., 840 Memorial Drive, Third Floor, Cam — without international search report and to be republished bridge, MA 02139 (US). upon receipt of that report (Rule 48.2(g)) (54) Title: NUTRITIVE POLYPEPTIDES, FORMULATIONS AND METHODS FOR TREATING DISEASE AND IMPROVING MUSCLE HEALTH AND MAINTENANCE 5 1 i & _a T * < & o O0 QO FIG. 17 © (57) Abstract: Nutritive polypeptides are provided herein. Also provided are various other embodiments including pharmaceutical formulations containing the nutritive polypeptides, nucleic acids encoding the polypeptides, recombinant microorganisms that make the polypeptides, vectors for expressing the polypeptides, methods of making the polypeptides using recombinant microorganisms, o compositions that comprise the polypeptides, and methods of using the polypeptides to treat or prevent diseases, disorders and con ditions associated with muscle wasting, and of using the polypeptides to improve and maintain muscle health. NUTRITIVE POLYPEPTIDES, FORMULATIONS AND METHODS FOR TREATING DISEASE AND IMPROVING MUSCLE HEALTH AND MAINTENANCE CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Patent Application No. 61/769,429, filed February 26, 2013; U.S. Patent Application No. 61/769,444, filed February 26, 2013; and U.S. Patent Application No. 61/808,650, filed April 5, 2013; the entire disclosures of which are hereby incorporated by reference in their entirety for all purposes. [0002] This application is related to PCT/US20 13/032232, filed March 15, 2013, PCT/US20 13/032 180, filed March 15, 2013, PCT/US20 13/032225, filed March 15, 2013, PCT/US20 13/0322 18, filed March 15, 2013, PCT/US20 13/0322 12, filed March 15, 2013, PCT/US20 13/032206, filed March 15, 2013, and PCT/US20 13/03 8682, filed April 29, 2013; the entire disclosures of which are hereby incorporated by reference in their entirety for all purposes. INTRODUCTION [0003] Proteins, carbohydrates and lipids are the three macronutrients consumed by organisms to maintain health, as a direct energy input and as substrates for biosynthesis. In addition, dietary proteins contain essential amino acids which cannot be synthesized in the human body. The properties of a protein to elicit specific metabolic effects in an individual is termed "protein quality" (Milward et al 2008 Am J Clin Nutr. 87(5): 15765-15815.), therefore the protein quality requirements of individuals differ on the basis of such conditions as disease states, medications and physical activity. [0004] The maintenance of skeletal muscle mass is important for maintaining quality of life. Muscle atrophy, the decrease in skeletal muscle mass, can be caused by chronic diseases such as cancer, chronic inflammation, diabetes and heart failure and generally accepted to result in a poor prognosis. Skeletal muscle loss associated with advancing age, or sarcopenia, is a major cause of decrease health and function among older adults impacting strongly on independence and quality of life (International Working Group of Sarcopenia, 201 1). Loss of muscle mass is proximally caused by a rate of proteolysis in excess of protein synthesis in skeletal muscle tissue (Combaret at al 2009 Curr Opin Clin Nutr Metab Care 12(1): 37-41). [0005] Dietary proteins and free essential amino acids have been investigated as methods for increasing the rate of muscle protein synthesis in elderly populations. Supplementation with leucine, an amino acid associated with muscle protein synthesis, or other essential amino acids alone may not be as effective as dietary protein consumption in stimulating muscle protein synthesis (Katsanos et al 2008 Nutr Res 28(10): 651-658, Magne et al 2012 J Physiol 590(8): 2035-2049, Magne et al 2013 PLOS One 8(8): e70130). While leucine is considered important for the stimulation of muscle protein synthesis, protein mixtures such as whey have been compared with free essential amino acids such as leucine in the capacity for sustaining muscle protein synthesis (Churchward-Venne et al, 2012 J Physiol 590(1 1): 2751-2765). [0006] The "mammalian target of rapamycin (mTOR)" is a protein kinase and a key regulator of cell growth, notably via protein synthesis. mTOR acts as a master regulator of cellular metabolism that nucleates two complexes, mTORCl and mTORC2, that have different kinase specificity and distinct protein partners. mTOR complex 1 (mTORCl) consists of regulatory associated protein of mTOR (raptor), mTOR associated protein LST8 homolog (mLST8, also known as GbL) and DEP domain containing mTOR-inter-acting protein (Deptor). The second complex, mTORC2, is characterized by association with RPTOR-independent compan-ion of mTOR (rictor), Sinl, GbL, and Deptor. The diverse combinations of mTOR and partners permit mTOR to have different modes of regulations for different downstream functions, which in turn regulate different cell functionality. The essential nature of mTOR's function is evident in the early embryonic lethality of mTOR knockouts, and the varying deficiencies of tissue specific mTOR knockouts. Studies have shown that muscle-specific inactivation of mTOR leads to severe myopathy, resulting in premature death. mTOR activity, and thus its regulation of cellular function, is known to be regulated by many different stimuli, including amino acids and their metabolites. [0007] mTOR drives protein synthesis across tissues. mTORCl mediated response to growth signaling is gated by amino acids. The localization of the response to lysosomes couples mTOR activation to muscle protein catabolism. mTORCl can be gated by EAAs, leucine, and glutamine. Amino acids must be present for any upstream signal, including growth factors, to activate mTORCl (Blommaart et al., 1995; Hara et al, 1998). More recently, it was discovered that amino acid-dependent activation of mTORCl requires the Rag GTPases (Kim et al., 2008; Sancak et al, 2008). Amino acids promote the loading of RagA/B with GTP, which allows the heterodimer to interact with the raptor component of mTORCl (Sancak et al., 2008). This interaction results in the translocation of mTORCl from the cytoplasm to the lysosomal surface, where the Rag GTPases dock on a multisubunit complex called Ragulator (Sancak et al., 2010). [0008] The effects of individual amino acids such as leucine on mTOR function vary, with the branched-chain amino acids (BCAA) known to be particularly potent. While different amino acids can activate mTOR to varying degrees, certain combinations of amino acids can potentiate mTOR activation. The exact specifications that activate mTOR depend on the specific mTOR complex being targeted, which will vary by the composition of mTOR interactors. Moreover, how these mTOR complexes are activated is influenced by the local intracellular environment (i.e., plasma membrane or lysosomal location), and by the nature of the cell itself (i.e., adipocyte or muscle). For example, studies have shown that leucine is a potent activator of the mTOR pathway, particularly in muscle cells. The optimal peptide for mTOR activation varies according to the nature of the targeted mTOR complex and the cellular context that it exists within. [0009] It has been observed that certain peptides, containing certain amino acids, combinations of amino acids, and peptide sequences containing certain amino acids and combinations thereof, can be selectively taken up by specific tissues. Previous studies have demonstrated that tissue distribution of circulating peptides is non-random and specific peptide sequences home to specific locales (Arap et al, 2002).