USOO8748569B2

(12) United States Patent (10) Patent N0.: US 8,748,569 B2 Stupp et a]. (45) Date of Patent: Jun. 10, 2014

(54) AND METHODS TO Berendsen, A Glimpae of the Holy Grail?, Science, 1998, 282, pp. ELECTROSTATICALLY CONTROL 642-643.* BIOACTIVITY OF THE IKVAV PEPTIDE Voet et al, Biochemistry, John Wiley & Sons Inc., 1995, pp. 235 EPITOPE 241.* Ngo et al, Computational Complexity, Protein Structure Protection, (75) Inventors: Samuel I. Stupp, Chicago, IL (US); and the Levinthal Paradox, 1994, pp. 491-494.* Bradley et al., Limits of Cooperativity in a Structurally Modular Joshua E. Goldberger, Columbus, OH Protein: Response of the Notch Ankyrin Domain to Analogous (US); Eric J. Berns, Chicago, IL (US) Alanine Substitutions in Each Repeat, J. M01. BIoL (2002) 324, 373-386.* (73) Assignee: Northwestern University, Evanston, IL Tysseling et al., “Self-assembling peptide promotes plas (Us) ticity of serotonergic ?bers following spinal cord injury,” J Neurosci Res, 2010, 88: 3161-3170. ( * ) Notice: Subject to any disclaimer, the term of this Tysseling-Mattiace et al., “Self-assembling nano?bers inhibit glial patent is extended or adjusted under 35 scar formation and promote axon elongation after spinal cord injury,” U.S.C. 154(b) by 0 days. JNeurosci, 2008, 28: 3814-3823. Wheeler et al., “Microcontact printing for precise control of nerve (21) Appl.No.: 13/442,210 growth in culture,” J Biomech Eng, 1999, 121: 73-78. Yamada et al., “Ile-Lys-Val-Ala-Val (IKVAV)-containing (22) Filed: Apr. 9, 2012 alphal chain form amyloid-like ?brils,” FEBS Lett, 2002, 530: 48-52. (65) Prior Publication Data Yeung et al., “Modulation of the growth and guidance of rat brain stem neurons using patterned extracellular matrix proteins,” US 2012/0294902 A1 Nov. 22,2012 Neurosci Lett, 2001, 301: 147-150. Yoshida et al., “Identi?cation of a heparin binding site and the bio logical activities of the laminin alphal chain carboxy-terminal globu Related US. Application Data lar domain,” JCell Physiol, 1999, 179: 18-28. Zhang et al., “Compatibility of neural stem cells With functionalized (60) Provisional application No. 61/473,593, ?led on Apr. self-assembling peptide scaffold in vitro,” Biotech Bioprocess Engi 8,2011. neering, 2010, 15: 545-551. Zou et al., “Growth of rat dorsal root ganglion neurons on a novel (51) 1111.0. self-assembling scaffold containing IKVAV sequence,” Mater Sci A61K38/02 (2006.01) Engineering: C, 2009, 29(7)o: 2099-2103. C07K2/00 (2006.01) Zustiak et al., “In?uence of cell-adhesive peptide ligands on poly A61K38/00 (2006.01) (ethylene glycol) physical, mechanical and transport prop A61K 9/51 (2006.01) erties,” Acta Biomater, 2010, 6: 3404-3414. B82Y5/00 (2011.01) Adams et al., “Growth cones turn and migrate up an immobilized gradient of the laminin IKVAV peptide,” J Neurobiol, 2005, 62: 134 (52) U.S.Cl. 147. CPC . A61K38/00 (2013.01); C07K2/00 (2013.01); Agheli et al., “Large area protein nanopatterning for biological appli C07K 2319/735 (2013.01); A61K 9/5169 cations,” Nano Lett, 2006, 6: 1165-1171. (2013.01); B82Y5/00 (2013.01); YIOS 977/795 Agius et al., “Antibodies directed against the beta l-integrin subunit (2013.01) and peptides containing the IKVAV sequence of laminin perturb USPC ...... 530/300; 530/345; 977/795 neurite outgrowth of peripheral neurons on immature spinal cord (58) Field of Classi?cation Search substrata,” Neurosci, 1996, 71:773-786. CPC A61K 38/00; A61K 9/0019; A61K 9/0024; A61K 9/5169; C07K 14/78; C07K 2319/735; (Continued) C07K 2/00; B82Y 5/00 See application ?le for complete search history. Primary Examiner * Julie Ha Assistant Examiner * Li Ni Komatsu (56) References Cited (74) Attorney, Agent, or Firm * Casimir Jones, SC. U.S. PATENT DOCUMENTS

2006/0247165 A1 11/2006 Stupp et al. (57) ABSTRACT OTHER PUBLICATIONS The present invention is directed to peptide amphiphile com pounds, compositions and methods of use, wherein nano?ber Cui et al, Spontaneous and X-rayiTriggered Crystallization at Long bundling or epitope aggregation is inhibited. In certain Range in Self-Assembling Filament Networks, Science, 2010, 327, embodiments, the peptide amphiphiles of the present inven pp. SSS-559* tion have increased solubility and reduced nano?ber bun Cui et al, Spontaneous and X-rayiTriggered Crystallization at Long Range in Self-Assembling Filament Networks, Science, 2010, 327, dling. The molecules may be used in pharmaceutical appli supporting materials, pp. 1-47.* cations, for example for in vivo administration to human Angeloni et al, Regeneration of the cavernous nerve by Sonic hedge patients, by increasing biological activity of the compositions hog using aligned peptide amphiphile nano?bers, Biomaterials, toward neurite outgrowth and nerve regeneration. 2011, 32, pp. 1091-1101.* Rudinger, Peptide Hormones, JA Parsons, Ed., 1976, pp. 1-7.* SIGMA, 2004, pp. 1-2.* 5 Claims, 17 Drawing Sheets US 8,748,569 B2 Page 2

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Nomizu et al., “Structure-activity study of a laminin alpha 1 chain Cui et al., “Cerebrum Repair with PHPMA Hydrogel Immobilized active peptide segment Ile-Lys-Val-Ala-Val (IKVAV),” FEBS Lett, with Neurite-Promoting Peptides in Traumatic Brain Injury of Adult 1995, 365: 227-231. Rat Model,” J Bioactive and Compatible Polymers, 2003, 18: 413 Ohga et al., “Design and activity of multifunctional ?brils using 432. receptor-speci?c small peptides,” Biomaterials, 2009, 30: 673 1 -673 8. Duque et al., “Immobilization of biomolecules to plasma polymer Patel et al., “Spatially controlled cell engineering on biodegradable ized penta?uorophenyl methacrylate,” Biomacromolecules, 2010 1 1 : polymer surfaces,” FASEB Journal, 1998, 12: 1447-1454. 2818-2823. 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$ 131 3 1% US 8,748,569 B2 1 2 PEPTIDE AMPHIPHILES AND METHODS TO form nanoscale aggregates having particular utility as bio ELECTROSTATICALLY CONTROL compatible scaffolds, more particularly in the area of tissue BIOACTIVITY OF THE IKVAV PEPTIDE engineering and regenerative medicine. EPITOPE Peptide amphiphiles (PAs) constitute a class of molecules that spontaneously self-assemble into a variety of nanostruc STATEMENT REGARDING FEDERAL tures, including spherical , cylindrical ?bers and rib FUNDING bons, and have shown promising therapeutic activity. PA molecules that form cylindrical ?ber-like nanostructures This invention was made with government support under (termed in the art “nano?bers”) have been found to be par grants number R01 EB003806 and 5 F32 EB007131 awarded ticularly useful in mimicking the biomechanical properties of by the National Institutes of Health and contract number the extracellular matrix. These PA molecules are composed W911NF-09-1-0044 awarded by the Department of Defense typically of four main segments: (1) a hydrophobic moiety, through the Defense Advanced Research Projects Agency commonly an acyl group derived from a fatty acid, that pro (DARPA). The government has certain rights in this inven motes molecular aggregation through hydrophobic collapse; tion. (2) a [3-sheet-forming peptide that promotes ?ber assembly; (3) a peptide segment that contains ionizable side-chain resi CROSS-REFERENCE TO RELATED dues; and (4) a peptide signaling moiety designed to interact APPLICATIONS with cellular receptors. These molecules self-assemble into high-aspect ratio nanostructures, forming gels in water at low The present invention claims the bene?t of US. Provi 20 concentrations when the charges on the ionic side chains are sional Patent Application Ser. No. 61/473,593, ?led Apr. 8, appropriately screened. Individual cylindrical nano?bers dis 2011, which is incorporated by reference in its entirety. play bioactive sequences on their surface in high density. However, aggregation of nano?bers into bundles reduces the FIELD OF THE INVENTION surface area for bioactive epitope presentation. Moreover, 25 some PA molecules have proven dif?cult to synthesize and/or The present invention relates generally to improved pep purify on a large scale. This can be due to the molecules’ tide amphiphiles (PAs) bearing the IKVAV (SEQ ID NO. 1) zwitterionic nature (i.e., carrying both positive and negative peptide sequence, peptide-amphiphile nano?bers and other charges), and their propensity to aggregate in solution due to nanostructures self-assembled therefrom and methods of the relative large proportion of non-polar amino acid residues. making and using same. In certain embodiments, the present 30 Peptide amphiphiles bearing the laminin-derived peptide invention relates to peptide amphiphiles incorporating three sequence IKVAV (SEQ ID NO. 1) have been reported in the or more ionizable side-chains disposed in such a manner as to prior art to selectively differentiate neural stem cells into modulate inter?ber interdigitation of an IKVAV (SEQ ID NO. neurons, to promote neurite outgrowth, and to suppress dif 1) epitope, or to promote surface presentation and biological ferentiation into astrocytes in vitro. Furthermore, IKVAV availability of the IKVAV (SEQ ID NO. 1) epitope, upon 35 bearing peptide amphiphiles have been reported to promote self-assembly of the peptide amphiphile into aggregated axonal regeneration, improve motor function, reduce apop structures. In certain embodiments, the present invention pro tosis and reduce astrogliosis in vivo in spinal cord lesions vides molecular designs of IKVAV (SEQ ID NO. 1) peptide following spinal cord injury (SCI) in rodents. In addition, amphiphiles that demonstrate greater ease of synthesis and therapeutics incorporating the IKVAV (SEQ ID NO. 1) pep puri?cation, are more soluble in salt solutions resembling 40 tide may be used to treat other neurodegenerative conditions extracellular ?uid, and exhibit surprisingly enhanced biologi such as Parkinson’s and Alzheimer’s disease, by promoting cal activity, including the promotion of neurite outgrowth, as regeneration of neurons from endogenous or exogenous neu compared with previously reported compounds. In certain ral stem cells. These compounds may also be used to mini embodiments, the peptide amphiphiles are used therapeuti mize tissue damage or promote repair or regenerate of brain cally (e.g., to treat injuries or diseases of the central nervous 45 tissue following cerebral trauma or stroke. system (CNS), including spinal cord injury (SCI)). The IKVAV (SEQ ID NO. 1) epitope has been shown to bind to at least two receptors, a 110 kDa laminin binding BACKGROUND OF THE INVENTION protein (LBPl 10/APP) and nucleolin, although the exact molecular arrangement of this binding and the signal trans Designing arti?cial extracellular matrices that effectively 50 duction pathways are not known from the prior art. The signal and direct cellular responses is essential for the cre IKVAV (SEQ ID NO. 1) peptide contains four hydrophobic ation of new regenerative medicine therapies. However, the amino acids with a strong propensity to form beta-sheet sec preparation of any synthetic material with structure on the ondary and tertiary structures. nanoscale that mimics natural tissue is a challenging problem. Peptides containing this epitope are reported to form amy One approach has been to prepare molecules that spontane 55 loid-like ?brils. If the IKVAV (SEQ ID NO. 1) peptide epitope ously assemble into ?bers similar in morphology to the pro is hydrogen bonded in a rigid beta-sheet conformation with teins and proteoglycans that compose the natural extracellu neighboring epitopes, this will likely reduce its ability to bind lar matrix. In contrast to most synthetic biopolymers, the use to target cellular receptors. While the IKVAV (SEQ ID NO. 1) of small, self-assembling molecules facilitates control of peptide itself is known to promote neurite sprouting, in pre chemical and structural properties of these macromolecular 60 vious studies on IKVAV-bearing polymer scaffolds, enhanced assemblies. Injectable, self-assembling biomaterials capable neurite outgrowth and neuronal differentiation were not of forming scaffolds in situ around cells are promising thera observed, possibly due to reduced epitope presentation result peutic candidates because of their minimally invasive deliv ing from unfavorable aggregation. ery. The signaling ef?ciency of self-assembling bioactive Thus, there exists a technological need, unful?lled by the structures depends not only on molecular structure but also on 65 prior art, for an effective supramolecular strategy to control nano scale morphology. To that end, peptide amphiphiles have epitope presentation of hydrophobic bioactive peptides such been shown to self-assemble under suitable conditions to as IKVAV (SEQ ID NO. 1) to enhance signal transduction.