USOO6969702B2 (12) United States Patent (10) Patent No.: US 6,969,702 B2 Bertilsson et al. (45) Date of Patent: Nov. 29, 2005 (54) COMPOUNDS AND METHODS FOR OTHER PUBLICATIONS INCREASING NEUROGENESIS Jackowski, "Neural injury repair: hope for the future as (75) Inventors: Göran Bertilsson, Västerhaninge (SE); barriers to effective CNS regeneration become clearer,' Rikard Erlandsson, Sundyberg (SE); British Journal of Neurosurgery, (1995), 9, p. 303-317.* Jonas Frisen, Stockholm (SE); Anders Asanuma et al. (1996). Mol. Brain Res. 41: 210-215. Haegerstrand, Danderyd (SE); Jessica Cameron and McKay (1998). Current Opinion in Neurobiol. Heidrich, Arsta (SE); Nina Hellström, 8: 677-680. Södertälje (SE); Johan Haggblad, Cassidy and Frisen (2001). Nature 412: 690-691. Västgötagränd (SE); Katarina Jansson, Dinter et al. (1997). J. Mol. Med. 75: 95-102. Johanneshov (SE); Jarkko Kortesmaa, D'Sa and Duman (2002). Bipolar Disorders 4: 183–194. Stockholm (SE); Per Lindquist, Duman et al. (2001). 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Neuroscience 20: 7782-7789. patent is extended or adjusted under 35 Pahan et al. (1997). J. Biol. Chem. 272: 7786–7791. U.S.C. 154(b) by 116 days. Palmer (1985). Life Sciences 36: 1995–2006. Sakaki et al. (1997). J. Neurobiology 32: 62-68. Sugioka et al.(1996). J. Physiology 493: 855–863. (21) Appl. No.: 10/850,055 Sugioka et al. (1999). Int. J. Devil. Neuroscience 17: (22) Filed: May 19, 2004 163-172. Temple (2001). Nature 414: 112–117. (65) Prior Publication Data Vitolo et al. (2002). Proc. Natl. Acad. Sci. USA 99: US 2005/0009742 A1 Jan. 13, 2005 13217-13221. Wyttenbach et al. (2001). Human Mol. Genetics 10: Related U.S. Application Data 1829-1845. Yamashita et al. (1994). J. Neurobiology 25: 1144-1153. (63) Continuation-in-part of application No. 10/718,071, filed on Yamashita and Sugioka (1998). News Physiol. Sci. 13: Nov. 20, 2003. (60) Provisional application No. 60/427,912, filed on Nov. 20, 75-79. 2002. * cited by examiner (51) Int. Cl." ......................... A61N 37/18: A61K 38/00 Primary Examiner Karen Cochrane Carlson (52) U.S. Cl. .......................... 514/12; 530/307; 530/324 ASSistant Examiner Agnes Rooke (58) Field of Search ............................ 514/12; 530/307, (74) Attorney, Agent, or Firm Mintz Levin Cohn Ferris 530/324 Clovsky and Popeo PC (56) References Cited (57) ABSTRACT The invention is directed to methods of promoting neuro U.S. PATENT DOCUMENTS genesis by contacting neuronal tissue with neurogenesis 5,665,380 A * 9/1997 Wallach et al. ............. 424/450 increasing agents. Novel methods for treating neurological disorders using neurogenesis increasing agents are dis FOREIGN PATENT DOCUMENTS closed. WO WO OO/12099 3/2000 WO WO 01/85981 11/2001 16 Claims, 3 Drawing Sheets U.S. Patent Nov. 29, 2005 Sheet 1 of 3 US 6,969,702 B2 A. PACAP Mouse Human B. Cholera toxin C CT15. CT6 CTsh E. assists Mouse Human FIG. 1 U.S. Patent Nov. 29, 2005 Sheet 2 of 3 US 6,969,702 B2 Sub Ventricular Zone A Brdu + 30 cells/300am * p=0.007 20 ** p=0.004 10 O Exendin-4 Calcitonin vehicle B Dentate Gyrus BdU 7.5 cells/300m p=0.003) 5.0 p= 2.5 OO Fxendin-4 Calcitonin vehicle FIG. 2 U.S. Patent Nov. 29, 2005 Sheet 3 of 3 US 6,969,702 B2 Dose-response of Calcitonin - Calcitonin F.G. 3 US 6,969,702 B2 1 2 COMPOUNDS AND METHODS FOR by progenitor cells (Johe et al., 1996; Williams et al., 1997). INCREASING NEUROGENESIS Recently, it has been shown that indeed adult regenerated neurons are integrated into the existing brain circuitry, and RELATED APPLICATIONS contribute to ameliorating neurological deficits (Nakatomi et 5 al., 2002). Interestingly, observations have also shown that This application is a continuation in part of U.S. Ser. No. neurogenesis is occurring not only at the level of the 10/718,071, filed Nov. 20, 2003, which claims benefit of olfactory bulb and hippocampus. In this respect it has been U.S. Ser. No. 60/427,912, filed Nov. 20, 2002. Suggested by Zhao et al. that this process can also occur in the adult mouse Substantia nigra, opening up a new field of FIELD OF THE INVENTION investigation for the treatment of Parkinson's disease (Zhao The invention is directed to in vitro and in vivo methods et al., 2003). of modulating neurogenesis. Novel agents for increasing The ability to expand neural progenitors and manipulate intracellular levels of cAMP, Ca" and for modulating their cell fate has enormous implications for transplant neurogenesis are also provided. therapies for neurological diseases where specific cell types 15 are lost. Parkinson's disease (PD), for example, is charac BACKGROUND OF THE INVENTION terized by degeneration of dopaminergic neurons in the Neural stem cells (NSC) are a source for new neurons in substantia nigra. Previous transplantation treatments for PD the mammalian CNS. NSC are located within the ependymal patients have used fetal tissue taken from the Ventral mid and/or subventricular Zone (SVZ) lining the lateral ventricle brain at a time when Substantia nigra dopaminergic neurons (Doetsch et al., 1999; Johansson et al., 1999b) and in the are undergoing terminal differentiation (Herman and dentate gyrus of the hippocampal formation (Gage et al., Abrous, 1994). These cells have been grafted onto the 1998). Studies have revealed the potential for several addi striatum where they form Synaptic contacts with host striatal tional locations of NSC within the adult CNS (Palmer et al., neurons, their normal Synaptic target. This restores dopam 1999). Asymmetric division of NSC maintains their starting ine turnover and release to normal levels with Significant number, while generating a population of rapidly dividing 25 functional benefits to the patient (Herman and Abrous, 1994) (for review see Bjorklund and Lindvall, 2000). However, the precursor, or progenitor cells (Johansson et al., 1999b). The grafting of fetal tissue is limited by ethical considerations progenitor cells respond to a range of cues that dictate the and a lack of donor tissue. The expansion and manipulation extent of their proliferation and their fate, both in terms of of adult NSC can potentially provide a range of well differentiation and positioning. characterized cells for transplant-based Strategies for neu The NSC of the ventricular system in the adult are likely rodegenerative disease Such as PD. To this aim, the identi counterparts of the embryonic ventricular Zone stem cells fication of factors and pathways that govern the proliferation lining the neural tube. The progeny of these embryonic cells and differentiation of neural cell types is fundamentally migrate away to form the CNS as differentiated neurons and important. glia (Jacobson, 1991). NSC persist in the adult lateral 35 ventricle wall (LVW), generating neuronal progenitors that Studies have shown that intraventricular infusion of both migrate down the rostral migratory Stream to the olfactory EGF and basic FGF induces proliferation in the adult bulb. There, they differentiate into granule cells and peri ventricle wall cell population. In the case of EGF, extensive glomerular neurons (Lois and Alvarez-Buylla, 1993). Sub migration of progenitors into the neighboring striatal paren Stantial neuronal death occurs in the olfactory bulb, creating 40 chyma has been observed (Craig et al., 1996; Kuhn et al., a need for continuous replacement of lost neurons which is 1997). EGF increases differentiation into glial lineage and satisfied by the migrating progenitors derived from the LVW reduced the generation of neurons (Kuhn et al., 1997). Additionally, intraventricular infusion of BDNF in adult rats (Bieblet al., 2000). In addition, there are indications that lost increases the number of newly generated neurons in the neurons from other brain regions can be replaced by pro olfactory bulb and rostral migratory Stream, and in paren genitors from the LVW that differentiate into the phenotype 45 chymal Structures, including the Striatum, Septum, thalamus of the lost neurons with appropriate neuronal projections and and hypothalamus (Pencea et al., 2001). Thus, several stud Synapses with the correct target cell type (Snyder et al., ies have shown that the proliferation of progenitors within 1997; Magavi et al., 2000). the SVZ of the LVW can be stimulated and that their lineage In vitro cultivation techniques have been established to can be guided to neuronal or glial fates. Yet, the number of identify the external Signals involved in the regulation of 50 factors known to affect neurogenesis in Vivo is Small and NSC proliferation and differentiation (Johansson et al., their effects are adverse or limited. 1999b; Johansson et al., 1999a). The mitogens EGF and basic FGF allow cell culture expansion of neural progenitors BRIEF SUMMARY OF THE INVENTION isolated from the Ventricle wall and the hippocampus (McKay, 1997; Johansson et al., 1999a). These dividing 55 One embodiment of the invention is directed to a method progenitors remain in an undifferentiated State, and grow for modulating neurogenesis in neural tissue of a patient that into large clones of cells known as neuroSpheres.