WO2004105787A1

WO2004105787A1 - Methods of using combinations of egf-2 and egf-20 to treat central nervous system disorders

Info

Publication number: WO2004105787A1
Application number: PCT/IB2004/001758
Authority: WO
Inventors: Nobuyuki Itoh
Original assignee: The University Of Kyoto
Priority date: 2003-05-28
Filing date: 2004-05-28
Publication date: 2004-12-09

Abstract

Compositions and methods for treating central nervous system (CNS) disorders in a mammal are provided. Compositions comprise combinations of fibroblast growth factor-2(FGF-2) and fibroblast growth factor-20 (FGF-20) in therapeutically effective amounts to treat central nervous system disorders such as Parkinson's disease and other neurodegenerative disorders. Methods of therapy comprise co-administration of FGF-2 and FGF-20 to a mammal in need of treatment for a CNS disorder. The invention also relates to methods of promoting the growth, proliferation, differentiation, and/or survival of a CNS cell, particularly dopaminergic neurons, in vitro and in vivo.

Description

METHODS OF USING COMBINATIONS OF FGF-2 AND FGF-20 TO TREAT CENTRAL NERVOUS SYSTEM DISORDERS

FIELD OF THE INVENTION The invention relates to methods and pharmaceutical compositions for treating central nervous system disorders, particularly the administration of compositions that comprise combinations of fibroblast growth factor-2 (FGF-2) and fibroblast growth factor-20 (FGF-20) to treat central nervous system disorders such as Parkinson's disease and other neurodegenerative disorders. The invention also relates to methods for promoting the growth, proliferation, differentiation, and/or survival of a CNS cell, particularly dopaminergic neurons, in vitro and in vivo.

BACKGROUND OF THE INVENTION

The central nervous system (CNS) consists of the brain and spinal cord. Disorders of the CNS affect a large portion of the population and are frequently associated with chronic and progressive symptoms. CNS disorders comprise a variety of neurological and neuropsychiatric disorders, including neurodegenerative disorders, behavioral disorders, affective disorders, and cognitive disorders. These disorders have been linked to such potential causes as genetic predisposition, environmental triggers including allergies, adverse reactions to drugs, traumatic injuries, and cerebrovascular events such as aneurysms or strokes. In addition, many CNS disorders are idiopathic in nature. Many neurological conditions result from the loss of certain cell populations from the nervous system through disease or injury. The cells destroyed in these conditions are not intrinsically replaced. Such neurodegenerative CNS disorders include, but are not limited to, Parkinson's disease, Huntington's disease, Alzheimer's disease and related dementias, Amyotrophic Lateral Sclerosis, Down's syndrome, Korsakoff s disease, and epilepsy. Recent evidence demonstrates that neuronal replacement and partial reconstruction of neuronal circuitry is possible via cell transplantation therapies. Much of the initial work in the field used fetal-cell therapies. In recent years, however, it has become evident that the developing and even the adult mammalian nervous system contains a population of undifferentiated, multipotent, neural stem cells that display plastic properties that are advantageous for the design of more effective neural regenerative strategies for many of these neurological conditions.

For example, in Parkinson's disease, the neurons that degenerate comprise the dopaminergic neurons of the substantia nigra. Current cell transplantation strategies for patients with advanced Parkinson's disease comprise intrastriatal grafts of nigral dopaminergic neurons from 6- to 9-week-old human embryos. Clinical improvements develop gradually over the first 6-24 months after transplantation (Olanow et al. (1996) Trends Neurosci. 19:102-109 and Lindvall et al. (1999) Mov. Disord. 14:201- 205). Further, the first study of human fetus-to-adult striatal transplantation has recently been performed in three nondemented patients with moderately advanced Huntington's disease. Magnetic resonance imaging evaluation at one year documented graft survival and growth without displacement of surrounding tissue. All patients improved on some measure of cognitive function, although no uniform pattern was evident (Kopyov et al. (1998) J. Exp. Neurol. 149:97-108). See also, Date et al. (1997) J. Exp. Neurol. 147:10-17.

Neural stem cells have also been demonstrated to replace lost and dying cells and lost neural circuits in the degenerating CNS. For instance, treatment of mice with MPTP, a neurotoxin that selectively destroys dopaminergic cells in the substantia nigra, followed by grafting with a neural stem cell population, resulted in a reconstituted dopaminergic cell population composed of both donor and host cells. Similar studies in mice using a hypoxia-ischemic brain injury model showed transplantation of neural stem cells enhanced the recovery of the damaged system (Park et al. (1999) J. Neurotrauma 16:675-687 and Park et al. (1997) Soc. Neurosci. Abst. 23:346). Other treatment options for neurodegenerative disorders include medications that increase the availability of a specific neurotransmitter (e.g., administration of levodopa for Parkinson's disease). However, such treatments have shown limited efficacy for many patients and have been associated with a range of side effects (e.g, for levodopa: nausea, vomiting, loss of appetite, and dyskinesias).

Clearly, better methods of therapy for treating CNS disorders, particularly neurodegenerative disorders, are needed.

SUMMARY OF THE INVENTION Pharmaceutical compositions and methods for treating central nervous system (CNS) disorders, particularly Parkinson's disease and other neurodegenerative disorders, in a mammalian subject are provided. The pharmaceutical compositions comprise a therapeutically effective amount of fibroblast growth factor 2 (FGF-2) or biologically active variant thereof in combination with a therapeutically effective amount of fibroblast growth factor 20 (FGF-20) or biologically active variant thereof, and a pharmaceutically acceptable carrier. Such compositions when administered in accordance with the methods of the invention provide effective treatment for mammalian subjects suffering from a CNS disorder, particularly Parkinson's disease and other neurodegenerative disorders.

Methods for treating a mammalian subject for a central nervous system (CNS) disorder are provided. In one embodiment, the method comprises co-administration of fibroblast growth factor-2 (FGF-2) or biologically active variant thereof and fibroblast growth factor-20 (FGF-20) or biologically active variant thereof to the subject in need of treatment. Co-administration can be achieved using a single pharmaceutical composition comprising both of these growth factors, or two separate pharmaceutical compositions, each of which comprises one of these growth factors. This co-administration protocol can comprise administration of a single therapeutically effective dose of each of these agents; alternatively, multiple doses of these two agents can be administered as needed to treat the particular CNS disorder. The subject undergoing treatment with this method of co-administration optionally has received cell transplantation therapy within a region of the CNS. Combination therapy with FGF-2 or variant thereof and FGF-20 or variant thereof is more effective at promoting growth, proliferation, differentiation, and/or survival of a CNS cell

(which can be an existing CNS cell, a donor cell that has been transplanted within the CNS, or a cell that is derived from a transplanted donor cell) than is therapy with either agent alone. As a result, the methods of the invention allow for a greater therapeutic response as well as permit the use of lower concentrations of these two growth factors to reduce the risk of potential side effects.

In another embodiment, the method of treatment comprises culturing of neural progenitor cells, particularly those derived from embryonic stem cells, in the presence of a combination of FGF-2 or variant thereof and FGF-20 or variant thereof to obtain a population of differentiating neural progenitor cells, and subsequent transplantation of these cultured cells into the CNS of the subject in need of treatment for a CNS disorder. Optionally this procedure can be followed by co-administration of FGF-2 and FGF-20 or variants thereof to provide further enhancement of growth, proliferation, differentiation, and/or survival of these transplanted donor cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a time-course analysis for expression of neuroprogenitor markers on monkey embryonic stem (ES) cells. Monkey ES cells were plated on PA6 cells for the indicated period and then stained with anti-Musashi-1 (FITC) and anti- NCAM (Cy3) antibodies.

Figure 2 shows expression of neuro transmitter markers and differentiation markers in differentiated monkey ES cells. The spheres induced from monkey ES cells were plated on ornithin-laminin-coated slides; and 1 week later the differentiated spheres were stained with antibodies against TuJl, GFAP, Galactocerebroside C (GalC), Map2ab, GABA, glutamate (Glu), serotonin (Ser), or choline acetyltransferase (ChAT). Staining revealed that the spheres expressed the postmitotic neuronal marker TuJl, Map2ab, FGAP, and GalC (Figure 2A) and were immunopositive for expression of neurotransmitters GABA, ChAT, TH, serotonin, and glutamate (Figure 2B). These results indicate that the spheres were multipotential and consisted of neural stem cells.

Figure 3 shows the effect of FGF-20 on expansion of TH-positive cells. ES cell-derived spheres were cultured with in the presence of FGF-2; FGF-2 and EGF; FGF2 and FGF-20; FGF-20; and FGF-2, EGF, and FGF-20. Figure 4 shows the effect of neural progenitors transplanted into MPTP -treated monkeys. Figure 4A shows behavioral scores from ES cell-transplanted and sham- operated animals. The animals in which ES cell-driven neural progenitors were transplanted showed a significant improvement in neurological score when compared with sham-operated animals (*p<0.05, each value is given as the mean + SEM, n = 6 each). Figure 4B shows positron emission tomography (PET) of ES cell-transplanted and sham-operated monkeys. Mean Ki values obtained from the entire putamen are shown. After transplantation, improvement in 18F-fluorodopa binding was detected in ES cell-transplanted animals. However, no improvement in binding was detected in the sham-operated animals.

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to new alternatives for the treatment of central nervous system (CNS) disorders, particularly Parkinson's disease and other neurodegenerative disorders. In one embodiment, the invention provides a method of therapy wherein a mammalian subject in need of treatment for a CNS disorder is administered fibroblast growth factor-2 (FGF-2) or biologically active variant thereof in combination with administration of fibroblast growth factor-20 (FGF-20) or biologically active variant thereof. This subject optionally has undergone cell transplantation therapy within a region of the CNS, either prior to, simultaneously with, or following co-administration of these two growth factors. Co-administration of these two growth factors provides for a greater therapeutic benefit than can be obtained with either agent alone, or that can be achieved with conventional cell transplantation therapies. In another embodiment, the invention provides for the use of a combination of FGF-2 and FGF-20 to promote the growth, differentiation, and/or survival of a CNS cell, particularly dopaminergic neurons derived from embryonic stem cells, in preparation for transplantation into a target site within the CNS of a mammalian subject in need of treatment for a CNS disorder, particularly Parkinson's disease and other neurodegenerative disorders. Following transplantation, the subject can optionally be administered FGF-2 or variant thereof in combination with FGF-20 or variant thereof to supplement the growth, proliferation, differentiation, and/or survival of the transplanted cells. Pharmaceutical compositions comprising FGF-2 or variant thereof and FGF-20 or variant thereof for use in practicing the therapeutic methods of the invention are also provided herein. Although the essence of the present invention is described in terms of FGF-2 and FGF-20, it is to be understood that the various embodiments of the invention can be practiced using biologically active variants of these two growth factors, as defined elsewhere below. The methods of the present invention use a combination of FGF-2 and FGF- 20, or biologically active variants thereof, to treat mammalian subjects with a CNS disorder. FGF-2 and FGF-20 are members of the fibroblast growth factor (FGF) family of polypeptides, which comprises 23 members identified to date. The FGF family includes pluripotent growth factors that stimulate to varying extents fibroblasts, smooth muscle cells, epithelial cells, endothelial cells, myocytes, and neuronal cells. The FGF family members have numerous diagnostic and therapeutic uses.

Fibroblast growth factors bind to cell surface receptors that are ligand- stimulatable tyrosine kinases. Binding of these growth factors to their receptors leads to activation of intrinsic tyrosine kinase and signal transduction to downstream signaling cascades (Gerwins et al. (2000) Crit. Rev. Oncol. Hematol. 34(3):185-194).

One characteristic of all members of the FGF family is their ability to bind to heparin.

The various FGF molecules range in size from 15-23 kDa, and exhibit a broad range of biological activities in normal and malignant conditions including nerve cell adhesion and differentiation (Schubert et al. (1987) J. Cell Biol. 104:635-643), wound healing (U.S. Patent No. 5,439,818), mitogenic activity toward many mesodermal and ectodermal cell types, as trophic factors, as differentiation inducing or inhibiting factors (Clements et al. (1993) Oncogene 8: 1311-1316), and as angiogenic factors (Harada (1994) J. Clin. Invest. 94:623-630). Members of the FGF family have also been described as neurologic and/or neurotrophic agents (Hefti et al. (1989) Neurobiol. Aging 10:515-533; Cuevas and Gimenez-Gallego (1997) Neurol. Res. 19:254-256; Fisher and Finkelstein (1999) Cerebrovasc. Dis. 9:29-32; Mufson et al. (1999) Prog. Neurobiol. 57:451-484; Ay et al. (1999) Cerebrovasc. Dis. 9:131-135). The FGF-2 and FGF-20 to be administered can be from any animal species including, but not limited to, avian, canine, bovine, porcine, equine, and human. Generally, the FGF-2 and FGF-20 are from a mammalian species, preferably bovine or human, particularly bovine or human FGF-2 and human FGF-20 when the mammalian subject undergoing treatment is a human. The FGF-2 and FGF-20 may be in the native, recombinantly produced, or chemically synthesized forms as outlined below.

Fibroblast growth factor-2 (FGF-2, also known as basic FGF or bFGF), including recombinantly produced forms (rFGF-2), is a potent mitogen and angiogenic agent that has been recognized for its utility in the treatment of coronary artery disease (angina) and peripheral artery disease (claudication). FGF-2 expression is abundant in brain tissue (Gospodarowicz (1987) Methods Enzymol. 147:106-119) and exerts survival-enhancing effects on primary cultures from various regions of the brain (Walicke (1988) J. Neurosci. 8:2618-2627). FGF-2 is preferentially expressed in neurons in restricted regions including the cingulated cortex, indusium griseum, fasciola cinerea, and hippocampus, and in astrocytes in widespread regions of the brain (Emoto et al. (1989) Growth Factors 2:21-29; Woodward et al. (1992) J. Neurosci. 12:142-152). As disclosed herein, FGF-2 also has the ability to synergistically promote growth, proliferation, differentiation, and/or survival of a cell in the CNS, particularly dopaminergic neurons, when administered in combination with FGF-20.

Human and bovine FGF-2 are described in U.S. Patent No. 5,439,818 and U.S. Patent No. 5,155,214, respectively. The cDNA and amino acid sequences of native human FGF-2 (hFGF-2) are shown in SEQ ID NO: 1 and SEQ ID NO:2, respectively. The cDNA and amino acid sequences of bovine FGF-2 are shown in SEQ ID NO: 3 and SEQ ID NO:4, respectively.

The human and bovine FGF-2 shown in SEQ ID NO:2 and SEQ ID NO:4, respectively, represent the 146-amino-acid forms of these proteins. Both the human and bovine 146-amino-acid forms of FGF-2 are initially synthesized in vivo as polypeptides having 155 amino acids (Abraham et al. (1986) EMBO J. 5(10):2523- 2528; SEQ ID NO:6 of human origin; SEQ ID NO:8 of bovine origin). When compared to the full-length 155-residue FGF-2 molecules, the 146-residue FGF-2 molecules lack the first nine amino acid residues at the N-terminus of the corresponding full-length 155-residue human and bovine FGF-2 molecules (SEQ ID NO:6 and SEQ ID NO:8, respectively). The 155-residue FGF-2 of human or bovine origin, and biologically active variants thereof, can also be used in the methods of the present invention in the manner described for the human and bovine 146-residue FGF-2 molecules. The bovine FGF-2 set forth in SEQ ID NO:4 differs from human FGF-2 set forth in SEQ ID NO:2 in two residue positions. In particular, the amino acids at residue positions 1 12 and 128 of the bovine FGF-2 set forth in SEQ ID NO:4 are Ser and Pro, respectively, whereas in human FGF-2 (SEQ ID NO:2), they are Thr and Ser, respectively. For the 155-residue forms, these differences appear at residue positions 121 and 137 of SEQ ID NO:8 (FGF-2 of bovine origin) and SEQ ID NO:6 (FGF-2 of human origin).

The FGF-2 for use in the present invention may be derived from various mammalian tissues known to express the factor of interest, such as the brain and pituitary. FGF purification can be achieved by heparin-sepharose column chromatography as described in Gospodarowicz et al. (1984) Proc. Natl. Acad. Sci. USA 81 :6963-6967 or in U.S. Patent No. 5,310,883. Purification may also be achieved using β-cytodextin tetradeca sulfate affinity chromatography as described in Shing et al. (1990) Anal. Biochem. 185:108-111; all of which are herein incorporated by reference.

Alternatively, the FGF-2 can be recombinant FGF-2. By "recombinant FGF- 2" is intended FGF-2 having comparable biological activity to native- sequence FGF-2 and that has been prepared by recombinant DNA techniques, or mutationally altered FGF-2. In general, the gene coding for FGF-2 may be cloned and then expressed in transformed organisms, preferably a microorganism. The host organism may express the foreign gene to produce FGF-2 under expression conditions. Synthetic recombinant FGF-2 can also be made in eukaryotes, such as yeast or human cells. It is recognized that the 155-residue form of FGF-2 may exist as 153-155 residues, or mixtures thereof, depending upon the method of recombinant protein production (see U.S. Patent No. 5,143,829, herein incorporated by reference). Recombinant FGF-2 can be made as described in U.S. Patent No. 5,155,214, herein incorporated by reference. Methods of purifying recombinant FGF-2 to pharmaceutical quality can be found in, for example, U.S. Patent No. 4,956,455.

The term "human FGF-20" as used herein refers to a fibroblast growth factor family member that is highly expressed in dopaminergic neurons of the substantia nigra of brain (see International Publication No. WO 01/31008, herein incorporated by reference in its entirety). The cDNA and amino acid sequences of native human FGF-20 (hFGF-20) are shown in SEQ ID NO:9 and SEQ ID NO: 10, respectively. The cDNA and amino acid sequences of rat FGF-20 are shown in SEQ ID NO:l 1 and SEQ ID NO: 12, respectively. Human FGF-20 and rat FGF-20 share approximately

95% amino acid sequence identity. An alignment of selected members of the FGF family shows that FGF-20 is most closed related to FGF-9 and FGF- 16 (see Figures 1 and 2 of International Publication No. WO 01/31008, herein incorporated by reference). FGF-20 shares approximately 70% and 62% amino acid sequence identity with FGF-9 and FGF- 16, respectively. Human and rat FGF-20 have a conserved amino acid residue core (residues 62-197 of SEQ ID NO:10 and SEQ ID NO:12) with a strong hydrophobic region. Two cysteine residues that are well conserved in the FGF family are also conserved in these proteins (residues 71 and 137 of SEQ ID NO: 10 and SEQ ID NO: 12). In both human and rat FGF-20, the heparin binding site comprises residues 170-186 of SEQ ID NO: 10 and 12, respectively.

FGF-20 is expressed preferentially in the substantia nigra pars compacta region of the brain (the region involved in Parkinson's disease) but is weakly or not at all expressed in most other tissues (Ohmachi et al. (2000) Biochem. Biophys. Res. Comm. 277:355-360). Recombinant rat FGF-20, expressed using a baculovirus system, was able to enhance the survival of midbrain dopaminergic neurons in cell culture and to protect cells from glutamate-induced injury (Ohmachi et al. (2000), supra). This indicates a potential involvement in other neurodegenerative disorders, such as motor neuron disease, multiple sclerosis, muscular dystrophy, diabetic neuropathy, Parkinson's disease, Alzheimer's disease, Huntington's disease, Korsakoff s disease, Down's Syndrome, sequelae of traumatic central nervous system injury, sequelae of chronic epilepsy, sequelae of stroke, sequelae of ischemia, and the like. By "recombinant FGF-20" or "rFGF-20" is intended FGF-20 having comparable biological activity to native-sequence FGF-20 and which has been prepared by recombinant DNA techniques, or mutationally altered FGF-20. In general, the gene coding for FGF-20 is cloned and then expressed in transformed organisms, preferably a microorganism. The host organism expresses the foreign gene to produce FGF-20 under expression conditions. Synthetic recombinant FGF-20 can also be made in eukaryotes, such as yeast or human cells. Processes for growing, harvesting, disrupting, or extracting the FGF-20 from cells are substantially described in, for example, International Publication No. WO 01/31008, herein incorporated by reference in its entirety. Methods of Therapy

The methods of the present invention comprise co-administration of FGF-2 and FGF-20 to a mammalian subject in need of treatment for a CNS disorder. Though the following discussion refers to FGF-2 and FGF-20 as the therapeutic agents to be co-administered, it is equally applicable to the use of biologically active variants of either of these two growth factors in the methods of the invention, so long as they retain the desired biological activity of the parent molecule as described herein below. Co-administration of FGF-2 and FGF-20 potentiates the effectiveness of either FGF-2 or FGF-20 administered singly, so that the therapeutic response is improved with respect to that observed with administration of either of these agents alone, and can be achieved with lower dosages of FGF-2 or FGF-20 than are required when a subject undergoes the same therapeutic protocol with the exception of administering either of these agents in the absence of the other agent. By "central nervous system disorder" or "CNS disorder" is intended any disorder or disease that affects the brain and/or spinal cord, including, but not limited to, a neurodegenerative disorder, an affective disorder, or nerve damage resulting from a cerebrovascular disorder, injury or trauma, or infection of the CNS. The term "neurodegenerative disorder" is used for all patients with a CNS disorder characterized by progressive nervous system dysfunction including, but not limited to, motor neuron disease, multiple sclerosis, muscular dystrophy, diabetic neuropathy, Parkinson's disease, Alzheimer's disease, Huntington's disease, Korsakoff s disease, Down's Syndrome, sequelae of traumatic central nervous system injury, sequelae of chronic epilepsy, sequelae of stroke, and sequelae of ischemia. By "co-administration" is intended that both of these therapeutic agents are delivered to cells within one or more target sites of the CNS within a time frame that allows for their combined beneficial effect on cell growth, proliferation, differentiation, and/or survival to occur within the cells of a target site, and/or within cells adjacent to the target site. By "target site" is intended the CNS tissue or structure that comprises cells in need of enhancement of their growth, proliferation, differentiation, and or survival. Such target sites encompass a variety of CNS tissues and structures including, but not limited to, the olfactory bulbs; the anterior olfactory nucleus; the midbrain; the medulla; the pons; the cerebellum; the hippocampal formation; the diencephalon; the frontal, temporal, occipital, and parietal cortices; the cervical spinal cord; the brain stem; the basal forebrain; and the caudate/putamen. In one embodiment, the target site comprises cells within the basal ganglia, for example, within the striatum (i.e., the caudate and putamen), globus pallidus, substantia nigra, substantia innominate, ventral pallidum, nucleus basalis of Meynert, ventral tegmental area, and/or subthalamic nucleus. A target site can be any CNS tissue or structure that comprises cells whose aberrant biological activity and/or degeneration results from or contributes to the morpho logical and/or behavioral neurological symptoms of the CNS disorder for which the subject is undergoing treatment. A target site can comprise existing cells, i.e., those cells that have been produced in situ within the CNS of the mammal undergoing treatment and which are autologous (i.e., of native origin) to the mammal undergoing treatment. Alternatively, a target site can comprise cells that have been transplanted into the target site, for example, transplanted neuronal stem cells, neuronal progenitor cells, committed neural progenitors, neurons, glia, and combinations thereof, as part of a cell transplantation or cell replacement strategy. Such transplanted cells are referred to herein as "transplanted donor cells." It is recognized that the term "transplanted donor cells" encompasses the original population of transplanted cells as well as cells that are derived from the original population of transplanted cells. Furthermore, transplanted donor cells can be heterologous (i.e., derived from an individual other than the subject undergoing treatment) or can be autologous (i.e., derived from a tissue obtained from the subject undergoing treatment, manipulated ex vivo, and subsequently transplanted back into the target site of the CNS of the subject undergoing treatment). Thus, in one embodiment of the invention, therapeutically effective amounts of FGF-2 and FGF-20 are co-administered to a target site within the CNS of the subject in need of treatment for a CNS disorder, where the target site comprises transplanted donor cells. A donor cell can be derived from any source and at various stages of developmental differentiation so long as the effect of the combination of these two growth factors on growth, proliferation, differentiation, and/or survival of the transplanted donor cell is sufficient to prevent or reduce the morphological and/or behavioral neurological symptoms of the disorder being treated. Donor cells can be derived from any fetal or adult neural tissue, including tissue from the hippocampus, cerebellum, spinal cord, cortex (i.e., motor or somatosensory cortex), striatum, basal forebrain (cholenergic neurons), ventral mesencephalon (cells of the substantia nigra), and the locus ceruleus (neuroadrenaline cells of the central nervous system).

In a preferred embodiment, the donor cell is a neural progenitor cell derived from embryonic stem cells. Embryonic stem cells are clonal cell lines derived from the inner cell mass of developing blastocysts. Embryonic stem cells are multipotent in that they are characterized by their ability to undergo continuous cellular proliferation, to regenerate exact copies of themselves (self-renewal), to generate a large number of regional cellular progeny, and to elaborate new cells in response to injury or disease. As such, ES cells are capable of differentiating into a variety of lineages.

A "neural progenitor cell" is an undifferentiated cell that is derived from a neural stem cell and which has a more limited self-renewal capacity and a more restricted potential for development into various cell lineages. Under appropriate conditions, neural progenitor cells will differentiate into neuroblasts (neuron generating cells) or fibroblasts (glia generating cells), which are commited to a particular path of differentiation. The use of such multipotent neuronal cell lineages for transplantation is known in the art. See, for example, Snyder et al. (1992) Cell 68:33, where multipotent neuronal cell lines have been grafted into the rat cerebellum to form neurons and glial cells. See, also, Campell et al. (1995) Neuron 15:1259- 1273; Fishell et al. (1995) Development 121:803-812; and, Olsson et al. (1995) Eur. J. Neurosci. 10:71-85.

Methods of isolation and transplantation of various neural progenitor cells derived from different tissues at different developmental stages are known in the art and include, for example, striatum cortex (Winkler et al. (1998) Mol. Cell. Neurosci. 11 :99-l 16; Hammang et al. (1997) Exp. Neurol. 147:84-95); cortex (Brustle et al. (1998) Nat. Biotechnol. 16:1040-1044 and Sabate et al. (1995) Nat. Genet 9:256- 260); human telencephalon (Flax et al. (1998) Nature 392:18-24 and Vescovi et al. (1999) Neuron 11 :951-966); hippocampus (Gage et al. (1995) J. Neurobiol. 36:249- 266 and Suhonen et al. (1996) Nature 383:624-627); basal forebrain (Minger et al.

(1996) Exp. Neurol. 141 :12-24); ventral mesencephalon (Winkler et al. (1998) Mol.

Cell. Neurosci. 11 :99-116; Svendsen et al. (1996) Exp. Neurol 137:376-388;

Hammang et al. (1997) Exp. Neurol. 147:84-95; Studer et al. (1997) Nat. Neurosci. 1 :290-295; Milward et al. (1997) J. Neurosci. Res. 50:862-871); and subventricular zone (Milward et al. (1997) Milward et al. (1997) J. Neurosci. Res. 50:862-871). Each of these references is herein incorporated by reference. In addition, methods for the isolation of neural stem cell progeny and methods to promote their differentiation can also be found in U.S. Patent Nos. 6,071,889, 6,103,530, and 5,851,832; herein incorporated by reference in their entirety. Methods for the isolation and culturing of neuroblasts are provided in U.S. Patent No. 6,045,807, herein incorporated by reference.

Though the transplanted neural progenitor cells can be from any source, examples of which are given above, preferably the neural progenitor cells are derived from embryonic stem (ES) cells, particularly human embryonic stem cells. Methods for culturing ES cell-derived neural progenitor cells are known in the art. See, for example, Nishimura et al. (2003) Stem Cells 21 :171-180; Okabe et al. (1996) Mech. Dev. 59:89-102; Lee et al. (2000) Nat. Biotechnol. 18:675-679; Kawasaki et al. (2000) Neuron 28:31-40; and Kawasaki et al. (2002) Proc. Natl. Acad. Sci. USA

99:1580-1585; Thomson et al. (1998) Science 282:1145-1147; Thomson and Odorico

(2000) Trends Biotechnol. 18:53-57; Amit et al. (2000) Dev. Biol. 227:271-278; Schuldiner et al. (2000) Proc. Natl. Acad. Sci. USA 97:11307-11312; Odorico et al.

(2001) Stem Cells 19:193-204; Reubinoff et al. (2001) Nat. Biotechnol. 19:1134- 1140; Schuldiner et al. (2001) Brain Res. 913:201-205; and Zhang et al. (2001) Nat.

Biotechnol. 19:1129-1133; the contents of which are herein incorporated by reference in their entirety.

In one preferred embodiment, prior to transplantation, the ES cell-derived neural progenitor cells are cultured in vitro in the presence of a combination of an amount of FGF-2 and an amount of FGF-20 sufficient to increase the percentage of dopaminergic neurons within the cultured population of differentiating progenitor cells that are to be used in neural transplantation therapy. By "population of differentiating neural progenitor cells" is intended the population of cultured neural progenitor cells comprises cells that are positive for one or more neural cell lineage differentiation markers. Such markers include, but are not limited to, TuJl (antigen expressed on neurons), GFAP (antigen expressed in astrocytes), GalC (antigen expressed on oligodendrocytes), MapZab (antigen expressed on neurons), and TH

(antigen expressed on neurons), which are readily detected with the use of RT-PCR and monoclonal antibodies known in the art. See, for example, Nishimura et al. (2003) Stem Cell 21 :171-180 and the assays described in the Examples below. FGF- 20 promotes differentiation of dopaminergic neurons from cultured ES cell-derived neural progenitor cells and/or promotes survival of these differentiated dopaminergic neurons during culture, in a concentration-dependent manner (see the Examples disclosed herein below). This effect is maximal in the presence of FGF-2. Thus, a population of ES cell-derived neural progenitor cells cultured in a suitable medium that includes the presence of both FGF-2 and FGF-20 as noted herein below comprises a higher percentage of differentiated dopaminergic neurons than does a population of ES cell-derived neural progenitor cells cultured in the same medium in the absence of the combination of FGF-2 and FGF-20. By "suitable medium" is intended any standard culture medium known in the art for maintaining a population of ES cell-derived neural progenitor cells and/or for differentiating neural and/or neuronal cells. See, for example, the media disclosed in Kawasaki et al. (2000) Neuron. 28:31-40 and Kawasaki et al. (2002) Proc. Natl. Acad. Sci. USA 99:1580- 1585. Also see the media described in Examples 1-2 herein below.

Detection of increased numbers of dopaminergic neurons in the cultured ES cell-derived neural progenitor cells relative to a population of ES cell-derived neural progenitor cells cultured in the same culture medium except for the absence of the combination of FGF-2 and FGF-20, or variants thereof, can be accomplished by various assays, including those described herein below. Such assays include monitoring expression of differentiation markers, particularly expression of tyrosine hydroxylase (TH). The presence of TH-positive differentiated dopaminergic neurons in cultured neural progenitor cells can be detected using anti-TH monoclonal antibodies known in the art (see, for example, the Experimental section herein below) and RT-PCR to detect TH expression (see, for example, Nishimura et al. (2003) Stem Cells 21 :171-180, herein incorporated by reference).

In this preferred embodiment, the ES cell-derived neural progenitor cells are cultured in a suitable medium comprising FGF-20 or biologically active variant thereof in the range of about 1 picamole (pM) to about 50 nanomole (nM), including, for example, 1 pM, 5 pM, 10 pM, 25 pM, 50 pM, 75 pM, 100 pM, 150 pM, 200 pM,

500 pM, 750 pM, 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 30 nM, 40 nM, 50 nM, and other such values between about 1 pM to about 50 nM of FGF-20 or biologically active variant thereof, and comprising FGF-2 or biologically active variant thereof in the range of about 1 pM to about 50 nM, including, for example, 1 pM, 10 pM, 25 pM, 50 pM, 75 pM, 100 pM, 150 pM, 200 pM, 500 pM, 750 pM, 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 30 nM, 40 nM, 50 nM, and other such values between about 1 pM to about 50 nM of FGF-2 or biologically active variant thereof. In preferred embodiments, the ES cell-derived neural progenitor cells are cultured in a suitable medium comprising FGF-20 or biologically active variant thereof in the range of about 100 pM to about 10 nM, including, for example, 100 pM, 150 pM, 200 pM, 250 pM, 300 pM, 350 pM, 400 pM, 450 pM, 500 pM, 550 pM, 600 pM, 650 pM, 700 pM, 750 pM, 800 pM, 850 pM, 900 pM, 950 pM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, and other such values between about 100 pM to about 10 nM, and comprising FGF-2 or biologically active variant thereof in the range of about 50 pM to about 1 nM, including, for example, 50 pM, 75 pM, 100 pM, 150 pM, 200 pM, 250 pM, 300 pM, 350 pM, 400 pM, 450 pM, 500 pM, 550 pM, 600 pM, 650 pM, 700 pM, 750 pM, 800 pM, 850 pM, 900 pM, 950 pM, 1 nM, and other such values between about 50 pM and about 1 nM. In one embodiment, the suitable medium comprises about 1 nM to about 10 nM FGF-20 or biologically active variant thereof, and about 50 pM to about 1 nM of FGF-2 or biologically active variant thereof.

The presence of the combination of FGF-2 and FGF-20 or biologically active variants thereof in these amounts provide an improved method for obtaining dopaminergic neurons from ES cell-derived neural progenitor cells for subsequent use in cell transplantation therapies for subjects in need of treatment for a CNS disorder, particularly a neurodegenerative disorder. When a population of ES cell-derived neural progenitor cells is cultured in medium comprising FGF-2 and FGF-20, or biologically active variants thereof, in the manner set forth herein, the resulting cultured population of differentiating neural progenitor cells comprises at least 5%, 8%), 10%, 15%o, or 20% of the cells as dopaminergic neurons, preferably at least 25%), 30%), or 35%, and more preferably at least 40%>, 45%, or 50%> of the cells as dopaminergic neurons, depending upon the concentration of FGF-2 and FGF-20, or biologically active variants thereof, present in the culture medium. Such populations of differentiating neural progenitor cells are referred to herein as "enriched."

Populations of cultured ES cell-derived neural progenitor cells enriched in differentiated dopaminergic neurons are particularly suited for neural transplantation therapies for subjects suffering from Parkinson's disease, which is characterized by progressive degeneration of this class of neurons in the substantia nigra pars compacta and a concomitant reduction in striatal dopamine.

Where the subject undergoing treatment for a CNS disorder is a receipient of cell transplantation therapy within a region of the CNS, co-administration of FGF-2 or variant thereof and FGF-20 or variant thereof to this subject can promote the growth, proliferation, differentiation, and/or survival of the transplanted donor cells, and cells derived from these transplanted donor cells, as well as promoting the growth, proliferation, differentiation, and/or survival of existing cells within the target site of the CNS of the subject being treated. In such embodiments, co-administration of FGF-2 and FGF-20 or variants thereof can be initiated prior to, simultaneously with, or following the transplantation procedure, with subsequent doses of FGF-2 and FGF- 20 or variants thereof being co-administered as needed to achieve the desired therapeutic response with respect to the CNS disorder being treated. Thus, a subject with a CNS disorder can undergoing therapy with co- administration of therapeutically effective amounts of FGF-2 or variant thereof and FGF-20 or variant thereof to enhance or promote the growth, proliferation, differentation, and/or survival of existing CNS cells within a target site of the CNS, and/or transplanted donor cells, for example, transplanted neural progenitor cells, within a target site of the CNS. Where the CNS cells within the target site are transplanted donor cells, these transplanted donor cells can also have been cultured in the presence of a combination of FGF-2 and FGF-20, or variants thereof, to promote differentiation and/or survival of the differentiating donor cells prior to their transplantation, particularly when these donor cells are ES cell-derived neural progenitor cells and the preferred cell type to be transplanted is differentiated dopaminergic neurons.

The FGF-2 and FGF-20 can be co-administered simultaneously in a single pharmaceutical composition comprising both of these therapeutic agents, for example, the pharmaceutical compositions provided elsewhere herein. Alternatively, these therapeutic agents can be co-administered simultaneously in two separate pharmaceutical compositions, each comprising one of these growth factors, through the same or different routes of administration. When formulated as two separate pharmaceutical compositions, the FGF-2 and FGF-20 can also be co-administered sequentially through the same or different routes of administration. By the term "sequentially" is intended the initial administration of either FGF-2 or FGF-20 alone, followed immediately or at some specified time later by administration of the second of these therapeutic agents. Co-administration can comprise a single administration of the therapeutically effective amounts of the FGF-2 or variant thereof and the FGF- 20 or variant thereof. Alternatively, co-administration can comprise multiple administrations of the therapeutically effective amount of FGF-2 or variant thereof in combination with the therapeutically effective amount of FGF-20 or variant thereof Pharmaceutical compositions comprising FGF-2 and/or FGF-20 are co- administered to a mammal, such as a human subject, having a target site within the CNS in need of treatment so as to deliver a therapeutically effective amount of each of these therapeutic agents to the target site within the CNS. Co-administration of the pharmaceutical compositions comprising FGF-2 and/or FGF-20 to an area in the CNS of a mammal, such as a human subject, is accomplished by any acceptable route of administration. Therapeutically effective amounts of FGF-2 and FGF-20 may be administered intranasally, orally, intravenously, subcutaneously, transmucosally (including buccally, lingually, and sublingually), topically, transdermally, by inhalation, intrathecally, intracerebrally, or using any other acceptable route of administration as noted elsewhere herein below.

Therapeutically Effective Doses and Therapeutic Responses

Delivery of therapeutically effective amounts of FGF-2 and FGF-20 thereof may be obtained via administration of a pharmaceutical composition comprising a therapeutically effective dose of FGF-2 and/or FGF-20. By "therapeutically effective amount" or "dose" is meant the concentration of FGF-2, or a variant thereof, and FGF-20, or a variant thereof, that is sufficient to elicit the desired therapeutic effect, as described herein. Accordingly, a therapeutically effective amount or dose of FGF- 2 and FGF-20 or variants thereof is characterized by an improvement in clinical symptoms for the CNS disorder to be treated. As such, a therapeutically effective amount or dose can be assayed via a reduction in neural deficits associated with the CNS disorder being treated, and hence is characterized by an improvement in clinical symptoms for the CNS disorder to be treated. Methods to quantify the extent of neurologic damage and to determine if the CNS disorder has been treated are well known to those skilled in the art. Such methods include, but are not limited to, histological methods, molecular marker assays, and functional/behavior analysis. For example, enhanced functional activity of existing CNS cells, enhanced functional integration of transplanted neural progenitor cells, for example, dopaminergic neurons derived from embryonic stem cells, and/or enhanced function and repair of the surrounding neuronal tissue can be assayed by examining the restoration of various functions including cognitive, sensory, motor, and endocrine. Motor tests include those that quantitate rotational movement away from the degenerative side of the brain, and those that assay for balance, coordination, slowness of movement, rigidity, and tremors. Cognitive tests include memory tests and spatial learning. The specific assays used to determine treatment of a neurologic disease will vary depending on the disorder.

Desired biological activities beneficial to an improvement in clinical symptoms for the CNS disorder to be treated include, for example, potentiation of the survival and or proliferation of an existing CNS cell or a transplanted donor cell (e.g., a dopaminergic neuron derived from an embryonic stem cell); improvement in the capacity of a transplanted donor cell to establish synaptic connection with the host neurons; and/or instruction of the transplanted donor cell to commit to a specific neural lineage. Methods to assay such events are known in the art. For example, an improvement in the survival of existing CNS cells or of transplanted donor cells following the co-administration of the FGF-2 and FGF-20, or variants thereof, can be assayed using various non-invasive scans such as computerized axial tomography (CAT scan or CT scan), nuclear magnetic resonance or magnet resonance imaging (NMR or MRS) or positron emission tomography (PET) scans. Alternatively, relevant information about donor cell survival can be assayed post-mortem by microscopic examination of the region of donor cell transplantation. The region of donor cells can be identified, for example, by assaying for molecular markers specific to the donor cells or alternatively, by prior incorporation of tracer dyes. Such dyes include, for example, rhodamine- or flourescein-labeled microspheres, fast blue, or retrovirally introduced histochemical markers.

The therapeutically effective amounts or doses of FGF-2 and FGF-20, or variants thereof, will depend on many factors including, for example, the CNS disorder being treated, the type of existing CNS cell contributing to the CNS disorder, the type of donor cell transplanted into the mammal, and the responsiveness of the subject undergoing treatment. In the case of transplanted donor cells, it is further recognized that the therapeutically effective amounts of these two therapeutic agents will depend on the type of developmental regulation of the donor cell that is desired (i.e., potentiation of the survival and/or proliferation of an existing CNS cell or the transplanted donor cell; improvement of the capacity of the transplanted donor cell to establish synaptic connection with the host neurons; regulation of the developmental cues released by the transplanted donor cells; or improved function and repair of the surrounding neural tissue). Methods to determine efficacy and dosage are known to those skilled in the art.

For example, in Parkinson's disease, the neurons that degenerate are the dopaminergic neurons of the substantia nigra. Cell transplantation therapies for patients with advanced Parkinson's disease are known and include, for example, intrastriatal grafts of nigral dopaminergic neurons from 6- to 9-week-old human embryos (Olanow et al. (1996) Trends Neurosci. 19:102-109 and Lindvall et al. (1999) Mov. Disord. 14:201-205). Delivery of pharmacologically active FGF-2 to regions of the brain affected by Parkinson's disease (i.e., midbrain and substantia nigra) has been demonstrated. See, for example, International Publication Nos. WO 00/33813 and WO 00/33814; and copending U.S. Patent Application Serial Nos. 09/458,566, and 09/458,562, both of which are herein incorporated by reference. As used herein, an "effective amount" of FGF-2 and FGF-20, or variants thereof, for the treatment of Parkinson's disease using the co-administration methods of the present invention will be sufficient to reduce or lessen the clinical symptoms of Parkinson's disease. As such, an effective amount of the FGF-2 and FGF-20, or variants thereof, co-administered by the methods of the present invention decreases the degeneration of existing neuronal cells, particularly dopaminergic neurons of the substantia nigra, via enhancement of their survival, and/or promotes the growth, proliferation, differentiation, and/or survival of transplanted donor cells that have transplanted using cell replacement strategies performed in the art for the treatment of

Parkinson's disease. Accordingly, the methods of the invention enhance survival and/or improve clinical status of the treated subject in comparison to subjects treated with either of these therapeutic agents alone, or treated with cell transplantation therapy alone or in combination with administration of only one of these therapeutic agents. Improvement in clinical status for Parkinson's disease includes, for example, improvement in the ventral mesencephalic graft efficacy in terms of apomorphine- induced rotational decrease, an increase in the density of striatal reinnervation, and an enhancement in neuronal survival (Tornqvist et al. (2000) Exp. Neurol. 164:130-138). Specific assays for these clinical improvements include using positron emission tomography (PET); normalization of dopamine synthesis and storage as assessed by striatal ¹⁸fluorodopa uptake; and spontaneous and drug-induced dopamine release as measured as dopamine D₂ receptor occupancy in the grafted putamen. See, for example, Piccini et al. (1999) Nat. Neurosci. 2 : 1137- 1140, herein incorporated by reference. Such assays can be readily used by one skilled in the art to determine the dosage range for the combined administration of FGF-2 and FGF-20, or variants thereof, for the effective treatment of Parkinson's disease.

Huntington's disease is characterized by progressive neurodegeneration, particularly in the striatum and cortex, which induces severe impairments in both motor and cognitive functions. Current cell transplantation therapies replace inhibitor connections from the striatum to other structures such as the globus pallidus through the implantation of striatal precursor cells. Delivery of pharmacologically active FGF-2 to regions of the brain that are affected by Huntington's disease (i.e., caudate- putamen, thalamus, dincephalon, cerebellum, and frontal cortex) has been demonstrated. See, for example, International Publication Nos. WO 00/33813 and WO 00/33814; and copending U.S. Patent Application Serial Nos. 09/458,566, and 09/458,562, both of which are herein incorporated by reference.

As used herein, an "effective amount" of FGF-2 and FGF-20, or variants thereof, for the treatment of Huntington's disease using the co-administration methods of the present invention will be sufficient to reduce or lessen the clinical symptoms of Huntington's disease. Thus, an effective amount of FGF-2 and FGF-20, or variants thereof, co-administered by the methods of the present invention decreases the degeneration of existing neuronal cells, particularly in the striatum and cortex, via enhancement of their survival, and/or promotes the growth, proliferation, differentiation, and/or survival of transplanted donor cells that have transplanted using cell replacement strategies commonly performed in the art for the treatment of

Huntington's disease. As such, the methods of the invention enhance survival and/or improve clinical status of the treated subject in comparison to subjects treated with either of these therapeutic agents alone, or treated with cell transplantation therapy alone or in combination with administration of only one of these therapeutic agents. Improvement in clinical status includes, for example, disinhibition of pallidal output, reduced locomotor hyperactivity, recovery of complex motor and cognitive behavior, and restitution of new habit-learning systems in the lesioned striatum. See, for example, Bjorklund et al. (1994) Functional Neural Transplantation (Raven, New York), pp.157-195; Dunnett et al. (1995) Behav. Brain Res. 66:133-142; Kendall et al. (1998) Nat. Med. 4:727-729; Palfi et al. (1998) Nat. Med. 4:963-966; Brasted et al. (1999) Proc. Natl. Acad. Sci. USA 96:10524-10529; and Wictorin et al. (1992) Prog. Neurobiol. 38:611-639; all of which are herein incorporated by reference. Such assays can be readily used by one skilled in the art to determine the dosage range for the combined administration of FGF-2 or a variant thereof and FGF-20 or a variant thereof for the effective treatment of Huntington's disease. Ischemic damage to the CNS, which can occur within the brain and/or spinal cord regions, can result from, for example, cardiac arrest or coronary artery occlusion, cerebral artery occlusion or stroke, and traumatic injury. Neural circuits of the CNS damaged following an ischemic event have been reconstructed using various cell transplantation therapies. For instance, for focal ischemia events, implantation of embryonic striatum into the damaged striatum (Hodges et al. (1994) Functional Neural Transplantation (Raven, New York), pp. 347-386) and implantation of neurons derived from a human teratocarcinoma cell line (Borlongan et al. (1998) Exp. Neurol. 149:310-321 and Borlongan et al. (1998) Neuroreport 9:3703-3709) have been performed. See also, for example, Hodges et al. (1996) Neurosci. 72:959-988, Sorensen et al. (1996) Exp. Neurol. 138:227-235, and Sinden et al. (1997) Neurosci. 81:599-608.

As used herein, an "effective amount" of FGF-2 or variant thereof and FGF-20 or variant thereof for the treatment of ischemic injury will be sufficient to reduce or lessen the clinical symptoms of the ischemic event. As such, an effective amount of the FGF-2 and FGF-20, or variants thereof, when co-administered by the methods of the present invention, will increase survival of CNS cells in and around the region of ischemia and/or and/or promotes the growth, proliferation, differentiation, and/or survival of transplanted donor cells that have been transplanted using cell transplantation therapies commonly performed in the art for the treatment of an ischemic injury.

Improvement in clinical status includes, for example, a reduction in infarct size, edema, and/or neurologic deficits (i.e., improved recovery of motor, sensory, vestibulomoter, and/or somatosensory function). Improvements further encompass a reduction in neural deficits, and hence improved recovery of motor, sensory, vestibulomoter, and/or somatosensory function.

Methods to determine if an ischemic event has been treated, particularly with regard to reduction of ischemic damage including infarct size, edema, and development of neural deficits, are well known to those skilled in the art. For example, after ischemic injury, there is a significant increase in the density of omega 3 (peripheral-type benzodiazepine) binding sites (Benazodes et al. (1990) Brain Res. 522:275-289). Methods to detect omega 3 sites are known and can be used to determine the extent of ischemic damage. See for example, Gotti et al. (1990) Brain Res. 522:290-307 and references cited therein. Alternatively, Growth Associated Protein-43 (GAP-43) can be used as a marker for new axonal growth following an ischemic event. See, for example, Stroemer et al. (1995) Stroke 26:2135-2144, and Vaudano et al. (1995) J. Neurosci 15:3594-3611. The therapeutic effect may also be measured by improved motor skills, cognitive function, sensory perception, speech and/or a decrease in the propensity to seizure in the mammal undergoing treatment. Such functional/behavior tests used to assess sensorimotor and reflex function are described in, for example, Bederson et al. (1986) Stroke 17:472-476, DeRyck et al. (1992) Brain Res. 573:44-60, Markgraf et al. (1992) Brain Res. 575:238-246, Alexis et al. (1995) Stroke 26:2338-2346. Enhancement of neuronal survival may also be measured using the Scandinavian Stroke Scale (SSS) or the Barthel Index. Such assays can be readily used by one skilled in the art to determine the dosage range for the combined administration of FGF-2 or a variant thereof and FGF-20 or a variant thereof for the effective treatment of an ischemic event.

As used herein, an "effective amount" of FGF-2 or variant thereof and FGF-20 or variant thereof for the treatment of CNS disorders, particularly neurodegenerative disorders, will be sufficient to reduce or lessen the clinical symptoms of such disorders. As such, an effective amount of the FGF-2 and FGF-20, or variants thereof factor, when co-administered by the methods of the present invention, will decrease degeneration of CNS cells at the target site of interest and/or promotes the growth, proliferation, differentiation, and/or survival of transplanted donor cells that have been transplanted using cell transplantation therapies commonly performed in the art for the treatment of CNS disorders, particularly neurodegenerative disorders. It will be understood that the total daily usage of FGF-2 or variant thereof and FGF-20 or variant thereof will be decided by the attending physician within the scope of sound medical judgment regarding the neurodegenerative disorder to be treated. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the CNS disorder, particularly neurodegenerative disorder, being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coinciding with FGF-2 or variant thereof and FGF-20 or variant thereof; and like factors well known in the medical arts.

Routes of Administration

As previously noted, the pharmaceutical composition comprising the FGF-2 and/or FGF-20, or variants thereof, can be administered using any acceptable route of administration so long as a therapeutically effective amount of both of these agents is delivered to the target site within the CNS of the subject in need of treatment. Where these agents are administered as two pharmaceutical compositions, the same or different routes of administration can be used, so long as these two therapeutic agents are being "co-administered" such that delivery of these two agents to the target site within the CNS is in a time frame that allows for their combined beneficial effect on cell growth, proliferation, differentiation, and/or survival to occur within the cells of a target site, and/or within cells adjacent to the target site. Acceptable routes of administration include, but are not limited to, any form of parenteral drug delivery, topical administration, oral administration, inhalation, nasal administration, and the like.

By the term "parenteral" drug delivery is meant delivery by passage of a drug into the blood stream without first having to pass through the alimentary canal or digestive tract. Parenteral drug delivery can be achieved with subcutaneous (i.e., administration under the skin), intramuscular (i.e., administration into muscle tissue), intradermal (i.e., administration into the skin), intravenous, or transdermal (i.e., delivery of a drug by passage of the drug through the skin and into the bloodstream) administration.

Another acceptable form of parenteral drug delivery is transmucosal, i.e., administration of a drug to a mucosal surface of an individual so that the drug passes through the mucosal tissue and into the individual's blood stream. Transmucosal drug delivery may be accomplished by administering the therapeutic agent to the buccal, transbuccal, lingual, sublingual, or nasal (i.e., with intranasal administration) mucosal surface.

The term "topical administration" is used in its conventional sense to mean delivery of a topical drug or pharmacologically active agent to the skin or mucosa. The term "oral administration" is used in its conventional sense to mean delivery of a drug through the mouth and ingestion through the stomach and digestive tract. The term "inhalation administration" is used in its conventional sense to mean delivery of an aerosolized form of the drug by passage through the nose or mouth during inhalation and passage of the drug through the walls of the lungs.

When using the foregoing routes of administration, delivery of these two therapeutic agents to one or more target sites within the CNS is accomplished via passage into the bloodstream. Because fibroblast growth factors do not readily cross the blood-brain barrier, when these routes of administration are to be used these two growth factors can be formulated with various agents that promote penetration or transport across the blood-brain barrier. Such agents include, but are not limited to, an antibody to the transferrin receptor (see, for example, Friden et al. (1995) Science 259:373-377; Song et al. (2002) J. Pharmacol. Exp. Ther. 301(2):605-610) and other such strategies for facilitating movement of peptide therapeutics across the blood- brain barrier (see, for example, Pardridge (2001) Jpn. J. Pharmacol. 87(2):97-103 and Bickel (2001) Adv. Drug. Deliv. Rev. 46(l-3):247-279). Alternatively, delivery of either or both of these two FGF family members directly into the CNS can be accomplished by administering the pharmaceutical composition(s) comprising these two growth factors directly to a tissue innervated by the trigeminal nerve and/or the olfactory nerve. Administration of FGF-2 and/or FGF- 20, or variants thereof, directly to a tissue innervated by the trigeminal nerve and/or the olfactory nerve allows for the transport of these administered agents via the trigeminal and or olfactory nerve pathways into a variety of CNS structures including, for example, the olfactory bulbs; the anterior olfactory nucleus; the midbrain; the medulla; the pons; the cerebellum; the hippocampal formation; the diencephalon; the frontal, temporal, occipital, and parietal cortices; the cervical spinal cord; the brain stem; the basal forebrain; and the caudate/putamen. Although the FGF-2 and FGF-20, or variants thereof, that are administered to tissues innervated by the trigeminal and/or olfactory nerve may be absorbed into the bloodstream as well as into these neural pathways, the FGF-2 and FGF-20, or variants thereof, preferably provides minimal effects systemically. In addition, the method of administration can provide for delivery of a more concentrated level of the FGF-2 and FGF-20, or variants thereof, to cells of the CNS as the FGF-2 and FGF-20, or variants thereof, do not become diluted in fluids present in the bloodstream. Methods for delivering various agents to the CNS via the trigeminal nerve and/or the olfactory nerve pathways can be found in, for example, International Publication Nos. WO 00/33813 and WO 00/33814; and copending U.S. Patent Application Serial Nos. 09/458,566, and 09/458,562, both of which are herein incorporated by reference.

In this manner, an effective amount of the FGF-2 and FGF-20 can be administered to one or both nasal cavities of the mammalian subject undergoing treatment or can be administered to a tissue that is innervated by the trigeminal nerve and which resides outside of the nasal cavity (referred to herein as extranasal administration). Administration in this manner obviates the obstacle of the blood- brain barrier and allows for more efficient delivery of these two growth factors to the target site within the CNS. Suitable tissues innervated by the trigeminal nerve include the nasal cavity tissue, particularly within the upper one-third of the nasal cavity, a conjunctiva, an oral tissue, or a skin tissue. When administering to the conjunctiva, a preferred tissue to be administered to is the mucosa of the lower or upper eyelid. Examples of suitable oral tissues include, but are not limited to, sublingual, a gingiva tissue, the anterior two-thirds of the tongue, the mucosa of a cheek, and the mucosa of the upper or lower lip. Examples of suitable skin tissues include, but are not limited to, skin of the face, the forehead, an upper eyelid, a lower eyelid, a dorsum of the nose, a side of the nose, an upper lip, a cheek, the chin, a scalp, or a combination thereof. For a more thorough discussion of transport of neuro trophic agents to the CNS along the trigeminal and/or olfactory neural pathways, see International Publication Nos. WO 00/33813 and WO 00/33814; and copending U.S. Patent Application Serial Nos. 09/458,566, and 09/458,562, both of which are herein incorporated by reference.

Dosing Regimens

It should be apparent to a person skilled in the art that variations may be acceptable with respect to the therapeutically effective dose and frequency of the administration of the FGF-2 and FGF-20 or variants thereof in accordance with the methods of the present invention. The amount of the FGF-2 and FGF-20 or variants thereof administered will be inversely correlated with the frequency of administration. Hence, an increase in the concentration of FGF-2 and FGF-20 or variants thereof in a single administered dose, or an increase in the mean residence time in the case of a sustained-release form of the FGF-2 and FGF-20 or variants thereof, generally will be coupled with a decrease in the frequency of administration.

It is recognized that a single dosage of the FGF-2 and FGF-20 or variants thereof may be administered over the course of several minutes, hours, days, or weeks. A single dose of the FGF-2 and FGF-20 or variants thereof may be sufficient. Alternatively, repeated doses may be given to a patient over the course of several hours, days, or weeks.

Further, the therapeutically effective amount or dose of a FGF-2 and FGF-20 or variants thereof and the frequency of administration will depend on multiple factors including, for example, the CNS disorder being treated, the severity of the CNS disorder being treated, and, if donor cells are utilized, the size of the tissue encompassed by the donor cells, and the type of donor cell transplanted into the mammal and on the type of developmental regulation of the donor cell that is desired (i.e., potentiate the survival and/or proliferation of the transplanted donor cell; improve the capacity of the transplanted donor cell to establish synaptic connection with the existing neurons; and influence the developmental cues released by the transplanted donor cells).

Some minor degree of experimentation may be required to determine the most effective dose and frequency of dose administration, this being well within the capability of one skilled in the art once apprised of the present disclosure. The co- adminstration methods of the present invention may be used with any mammal. Exemplary mammals include, but are not limited to rats, cats, dogs, horses, cows, sheep, pigs, and more preferably humans. For purposes of promoting growth, proliferation, differentiation, and/or survival of a transplanted donor cell, and thereby reducing or preventing the clinical manifestation of a CNS disorder being treated, co-administration of therapeutically effective doses of FGF-2 and FGF-20 or variants thereof may occur within minutes, hours, days, or even weeks of the initial transplantation of the donor cell. For example, the initial therapeutic dose may be administered within about 2 to 4 hours, within about 2 to 6 hours, within about 8 hours, within about 10 hours, about 15 hours, about 24 hours, within about 36 hours, about 48 hours, about 72 hours, or about 96 hours following transplantation of the donor cell. One or more additional doses may be administered within hours, days, or weeks following the initial dose. Furthermore, the mammal undergoing a cell transplantation therapy may be administered FGF-2 and FGF-20 or variants thereof within weeks, days, hours, or minutes prior to transplantation. Thus, for example, a mammal undergoing cell transplantation therapy can be administered therapeutically effective doses of FGF-2 and FGF-20 or variants thereof prior to, during, or following the surgical procedure. When administration is for the purpose of treatment, administration may be for either a prophylactic or therapeutic purpose. When provided prophylactically, these therapeutic agents are co-administered in advance of any symptom. The prophylactic administration of these therapeutic agents serves to prevent or attenuate any subsequent symptom. When provided therapeutically, these therapeutic agents are co- administered at (or shortly after) the onset of a symptom. The therapeutic co- administration of these two therapeutic agents serves to attenuate any actual symptom.

Pharmaceutical Compositions and Dosage Forms

The FGF-2 and FGF-20, or variants thereof, are formulated into pharmaceutical compositions for use in the methods of the present invention. In some embodiments of the invention, the FGF-2 and FGF-20, or variants thereof, are formulated as a single pharmaceutical composition to allow for co-administration of these agents at the same time via the same route of administration. Suitable compositions and dosage forms for use in the present invention include tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, transdermal patches, gels, powders, magmas, lozenges, creams, pastes, plasters, lotions, discs, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, and the like.

Suitable pharmaceutical compositions for carrying out the methods of co- administration of these two growth factors can be controlled-release, sustained- release, delayed-release, pulsatile-release, or immediate-release formulations, depending upon the desired objective and residence time for FGF-2 and FGF-20. The term "controlled release" is intended to refer to any drug-containing formulation in which release of the drug is not immediate, i.e., with a "controlled release" formulation, oral administration does not result in immediate release of the drug into an absorption pool. The term is used interchangeably with "non-immediate release" as defined in Remington: The Science and Practice of Pharmacy, Twentieth Ed. (Lippincott Williams & Wilkins, Philadelphia, Pennsylvania, 2000).

The "absorption pool" represents a solution of the drug administered at a particular absorption site, and k_r, k_a, and k_e are first-order rate constants for: 1) release of the drug from the formulation; 2) absorption; and 3) elimination, respectively. For immediate-release dosage forms, the rate constant for drug release k_r is far greater than the absorption rate constant k_a. For controlled-release formulations, the opposite is true, i.e., k_r <« k_a, such that the rate of release of drug from the dosage form is the rate-limiting step in the delivery of the drug to the target area. The term "controlled release" as used herein includes any non-immediate-release formulation, including but not limited to sustained-release, delayed-release, and pulsatile-release formulations. The term "sustained-release" is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that preferably, although not necessarily, results in substantially constant blood levels of a drug over an extended time period such as up to about 72 hours, about 66 hours, about 60 hours, about 54 hours, about 48 hours, about 42 hours, about 36 hours, about 30 hours, about 24 hours, about 18 hours, about 12 hours, about 10 hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour after drug administration. The term "delayed-release" is used in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that preferably, although not necessarily, includes a delay of up to about 10 minutes, about 20 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours.

The term "pulsatile-release" is used in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration. The term "immediate release" is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.

Many methods of preparation of a sustained-release formulation are known in the art and are disclosed in Remington: The Science and Practice of Pharmacy (2000), supra. Generally, the FGF-2 and/or FGF-20 or variants thereof can be entrapped in semipermeable matrices of solid hydrophobic polymers. The matrices can be shaped into films or microcapsules. Examples of such matrices include, but are not limited to, polyesters, copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al. (1983) Biopolymers 22: 547-556), polylactides (U.S. Patent No. 3,773,919 and EP 58,481), polylactate polyglycolate (PLGA) such as polylactide-co-glycolide (see, for example, U.S. Patent Nos. 4,767,628 and 5,654,008), hydrogels (see, for example, Langer et α/. (1981) J. Biomed. Mater. Res. 15:167-277; Langer (1982) Chem. Tech. 72:98-105), non-degradable ethylene-vinyl acetate (e.g., ethylene vinyl acetate disks and poly(ethylene-co-vinyl acetate)), degradable lactic acid-glycolic acid copolyers such as the Lupron Depot™, poly-D-(-)-3-hydroxybutyric acid (EP 133,988), hyaluronic acid gels (see, for example, U.S. Patent No. 4,636,524), alginic acid suspensions, nanoparticles (see, for example, De et al. (2001) Artif Cells Blood Substit. Immobil. Biotech. 29:31-46; Venugopalan et al. (2001) Pharmazie 56:217- 219; and Zhang et al. (2001) Ace. Chem. Res. 34:249-256; all of which are herein incorporated by reference), and the like. Suitable microcapsules can also include hydroxymethylcellulose or gelatin- microcapsules and polymethyl methacrylate microcapsules prepared by coacervation techniques or by interfacial polymerization. See International Publication No. WO

99/24061, entitled "Method for Producing Sustained-Release Formulations," wherein proteins are encapsulated in PLGA microspheres, herein incorporated by reference. In addition, microemulsions or colloidal drug delivery systems such as liposomes and albumin microspheres, may also be used. See Remington: The Science and Practice of Pharmacy (2000), supra. Other sustained-release compositions employ a bioadhesive to retain the pharmacologically active agent at the site of administration. Other pharmaceutical compositions that may be useful in administering a FGF-2 and/or FGF-20, or variants thereof of interest by the methods of the present invention include Captisol.

It is recognized that the total amount of FGF-2 and FGF-20 or variants thereof administered as a unit dose to a particular tissue will depend upon the type of pharmaceutical composition being administered, that is whether the composition is in the form of, for example, a solution, a suspension, an emulsion, or a sustained-release formulation. For example, where the pharmaceutical composition comprising a therapeutically effective amount of the FGF-2 and/or FGF-20 or variants thereof is a sustained-release formulation, the FGF-2 and FGF-20 or variants thereof are administered at a higher concentration. Those of ordinary skill in the art can readily deduce suitable formulations involving these compositions and dosage forms, including those formulations as described herein below.

Parenteral Administration

Parenteral administration, if used, is generally characterized by injection, including intramuscular, intraperitoneal, intravenous (IV), and subcutaneous injection. Injectable formulations can be prepared in conventional forms, either as liquid solutions or suspensions; solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Preferably, sterile injectable suspensions are formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable formulation may also be a sterile injectable solution or a suspension in a nontoxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. A more recently revised approach for parenteral administration involves use of a slow-release or sustained-release system (see, e.g., U.S. Pat. No. 3,710,795). Needle-free subcutaneous administration, for example, to an extranasal tissue innervated by the trigeminal nerve, may be accomplished by use of a device that employs a supersonic gas jet as a power source to accelerate an agent that is formulated as a powder or a microparticle into the skin. The characteristics of such a delivery method will be determined by the properties of the particle, the formulation of the agent and the gas dynamics of the delivery device. Similarly, the subcutaneous delivery of an aqueous composition can be accomplished in a needle-free manner by employing a gas-spring powered hand held device to produce a high force jet of fluid capable of penetrating the skin.

Transdermal Administration

The FGF-2 and FGF-20 may also be administered through the skin or mucosal tissue using conventional transdermal drug delivery systems, wherein the agent is contained within a laminated structure (typically referred to as a transdermal "patch") that serves as a drug delivery device to be affixed to the skin. Where the skin is innervated by the trigeminal nerve, this administration route has the advantage of promoting delivery directly to the CNS, thereby bypassing the blood-brain barrier (see International Publication No. WO 00/33814; and copending U.S. Patent Application Serial No. 09/458,562, herein incorporated by reference. Transdermal drug delivery may involve passive diffusion or it may be facilitated using electrotransport, e.g., iontophoresis. In a typical transdermal "patch," the drug composition is contained in a layer, or "reservoir," underlying an upper backing layer. The laminated structure may contain a single reservoir, or it may contain multiple reservoirs. In one type of patch, referred to as a "monolithic" system, the reservoir is comprised of a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like. Alternatively, the drug-containing reservoir and skin contact adhesive are separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, or it may be a liquid or hydrogel reservoir, or may take some other form. The backing layer in these laminates, which serves as the upper surface of the device, functions as the primary structural element of the laminated structure and provides the device with much of its flexibility. The material selected for the backing material should be selected so that it is substantially impermeable to the active agent and any other materials that are present, the backing is preferably made of a sheet or film of a flexible elastomeric material. Examples of polymers that are suitable for the backing layer include polyethylene, polypropylene, polyesters, and the like.

During storage and prior to use, the laminated structure includes a release liner. Immediately prior to use, this layer is removed from the device to expose the basal surface thereof, either the drug reservoir or a separate contact adhesive layer, so that the system may be affixed to the skin. The release liner should be made from a drug/vehicle impermeable material.

A skin patch formulated to mediate a sustained release of a composition can be employed for the transdermal delivery of FGF-2 and/or FGF-20 or variants thereof, to a tissue innervated by the trigeminal nerve. Where administered as a sustained- release formulation for transdermal delivery to the CNS, the skin patch will comprise a higher concentration of the FGF-2 and or FGF-20, or variants thereof.

Transdermal drug delivery systems may in addition contain a skin permeation enhancer. That is, because the inherent permeability of the skin to some drugs may be too low to allow therapeutic levels of the drug to pass through a reasonably sized area of unbroken skin, it is necessary to coadminister a skin permeation enhancer with such drugs. Suitable enhancers are well known in the art and include, for example, those enhancers listed below in transmucosal compositions.

Transmucosal Compositions and Dosage Forms

Transmucosal administration is carried out using any type of formulation or dosage unit suitable for application to mucosal tissue. For example, the FGF-2 and/or FGF-20 may be administered to the buccal mucosa in an adhesive tablet or patch, sublingually administered by placing a solid dosage form under the tongue, lingually administered by placing a solid dosage form on the tongue, administered nasally as droplets or a nasal spray, administered by inhalation of an aerosol formulation, a non- aerosol liquid formulation, or a dry powder, or the like. Preferred buccal dosage forms will typically comprise a therapeutically effective amount of the FGF-2 and or FGF-20, or variants thereof, and a bioerodible (hydrolyzable) polymeric carrier that may also serve to adhere the dosage form to the buccal mucosa. The buccal dosage unit is fabricated so as to erode over a predetermined time period, wherein drug delivery is provided essentially throughout. The time period is typically in the range of from about 1 hour to about 72 hours. Preferred buccal delivery preferably occurs over a time period of from about 2 hours to about 24 hours. Buccal drug delivery for short-term use should preferably occur over a time period of from about 2 hours to about 8 hours, more preferably over a time period of from about 3 hours to about 4 hours. As needed buccal drug delivery preferably will occur over a time period of from about 1 hour to about 12 hours, more preferably from about 2 hours to about 8 hours, most preferably from about 3 hours to about 6 hours. Sustained buccal drug delivery will preferably occur over a time period of from about 6 hours to about 72 hours, more preferably from about 12 hours to about 48 hours, most preferably from about 24 hours to about 48 hours. Buccal drug delivery, as will be appreciated by those skilled in the art, avoids the disadvantages encountered with oral drug administration, e.g., slow absorption, degradation of the active agent by fluids present in the gastrointestinal tract and/or first-pass inactivation in the liver. The "therapeutically effective amount" of the active agent (i.e., FGF-2 and/or

FGF-20) in the buccal dosage unit will of course depend on the potency of the FGF-2 or FGF-20 and the intended dosage, which, in turn, is dependent on the particular individual undergoing treatment, the specific indication, and the like. The buccal dosage unit will generally contain from about 1.0 wt. % to about 60 wt. %> active agent, preferably on the order of from about 1 wt. % to about 30 wt. %> active agent. With regard to the bioerodible (hydrolyzable) polymeric carrier, it will be appreciated that virtually any such carrier can be used, so long as the desired drug release profile is not compromised, and the carrier is compatible with FGF-2, FGF-20, and variants thereof, and any other components of the buccal dosage unit. Generally, the polymeric carrier comprises a hydrophilic (water-soluble and water-swellable) polymer that adheres to the wet surface of the buccal mucosa. Examples of polymeric carriers useful herein include acrylic acid polymers and co, e.g., those known as

"carbomers" (Carbopol®, which may be obtained from B. F. Goodrich, is one such polymer). Other suitable polymers include, but are not limited to: hydrolyzed polyvinylalcohol; polyethylene oxides (e.g., Sentry Polyox® water soluble resins, available from Union Carbide); polyacrylates (e.g., Gantrez®, which may be obtained from GAF); vinyl polymers and copolymers; polyvinylpyrrolidone; dextran; guar gum; pectins; starches; and cellulosic polymers such as hydroxypropyl methylcellulose, (e.g., Methocel®, which may be obtained from the Dow Chemical Company), hydroxypropyl cellulose (e.g., Klucel®, which may also be obtained from Dow), hydroxypropyl cellulose ethers (see, e.g., U.S. Pat. No. 4,704,285 to Alderman), hydroxyethyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, cellulose acetate phthalate, cellulose acetate butyrate, and the like.

Other components may also be incorporated into the buccal dosage forms described herein. The additional components include, but are not limited to, disintegrants, diluents, binders, lubricants, flavoring, colorants, preservatives, and the like. Examples of disintegrants that may be used include, but are not limited to, cross- linked polyvinylpyrrolidones, such as crospovidone (e.g., Polyplasdone® XL, which may be obtained from GAF), cross-linked carboxylic methylcelluloses, such as croscarmelose (e.g., Ac-di-sol®, which may be obtained from FMC), alginic acid, and sodium carboxymethyl starches (e.g., Explotab®, which may be obtained from Edward Medell Co., Inc.), methylcellulose, agar bentonite and alginic acid. Suitable diluents are those which are generally useful in pharmaceutical formulations prepared using compression techniques, e.g., dicalcium phosphate dihydrate (e.g., Di-Tab®, which may be obtained from Stauffer), sugars that have been processed by cocrystallization with dextrin (e.g., co-crystallized sucrose and dextrin such as Di- Pak®, which may be obtained from Amstar), calcium phosphate, cellulose, kaolin, mannitol, sodium chloride, dry starch, powdered sugar and the like. Binders, if used, are those that enhance adhesion. Examples of such binders include, but are not limited to, starch, gelatin and sugars such as sucrose, dextrose, molasses, and lactose. Particularly preferred lubricants are stearates and stearic acid, and an optimal lubricant is magnesium stearate.

Sublingual and lingual dosage forms include tablets, creams, ointments, lozenges, pastes, and any other solid dosage form where the active ingredient is admixed into a disintegrate matrix. The tablet, cream, ointment or paste for sublingual or lingual delivery comprises a therapeutically effective amount of the selected active agent and one or more conventional nontoxic carriers suitable for sublingual or lingual drug administration. The sublingual and lingual dosage forms of the present invention can be manufactured using conventional processes. The sublingual and lingual dosage units are fabricated to disintegrate rapidly. The time period for complete disintegration of the dosage unit is typically in the range of from about 10 seconds to about 30 minutes, and optimally is less than 5 minutes.

Other components may also be incorporated into the sublingual and lingual dosage forms described herein. The additional components include, but are not limited to binders, disintegrants, wetting agents, lubricants, and the like. Examples of binders that may be used include water, ethanol, polyvinylpyrrolidone; starch solution gelatin solution, and the like. Suitable disintegrants include dry starch, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic monoglyceride, lactose, and the like. Wetting agents, if used, include glycerin, starches, and the like. Particularly preferred lubricants are stearates and polyethylene glycol. Additional components that may be incorporated into sublingual and lingual dosage forms are known, or will be apparent, to those skilled in this art (See, e.g., Remington: The Science and Practice of Pharmacy (2000), supra).

Other preferred compositions for sublingual administration include, for example, a bioadhesive to retain the FGF-2 and/or FGF-20 or variants thereof sublingually; a spray, paint, or swab applied to the tongue; retaining a slow dissolving pill or lozenge under the tongue; or the like. Increased residence time increases the likelihood that the administered FGF-2 and/or FGF-20, or variants thereof, can be absorbed by the mucosal tissue and preferentially transported to the CNS along a nueral pathway that bypasses the obstacle presented by the blood-brain barrier.

Oral Dosage Forms

Oral dosage forms include tablets, capsules, caplets, solutions, suspensions and/or syrups, and may also comprise a plurality of granules, beads, powders or pellets that may or may not be encapsulated. Such dosage forms are prepared using conventional methods known to those in the field of pharmaceutical formulation and described in the pertinent texts, e.g., in Remington: The Science and Practice of Pharmacy (2000), supra). Tablets and capsules represent the most convenient oral dosage forms, in which case solid pharmaceutical carriers are employed.

Tablets may be manufactured using standard tablet processing procedures and equipment. One method for forming tablets is by direct compression of a powdered, crystalline or granular composition containing the active agent(s), alone or in combination with one or more carriers, additives, or the like. As an alternative to direct compression, tablets can be prepared using wet-granulation or dry-granulation processes. Tablets may also be molded rather than compressed, starting with a moist or otherwise tractable material; however, compression and granulation techniques are preferred.

In addition to the FGF-2 and/or FGF-20, or variants thereof, tablets prepared for oral administration using the method of the invention will generally contain other materials such as binders, diluents, lubricants, disintegrants, fillers, stabilizers, surfactants, preservatives, coloring agents, flavoring agents and the like. Binders are used to impart cohesive qualities to a tablet, and thus ensure that the tablet remains intact after compression. Suitable binder materials include, but are not limited to, starch (including corn starch and pregelatinized starch), gelatin, sugars (including sucrose, glucose, dextrose and lactose), polyethylene glycol, propylene glycol, waxes, and natural and synthetic gums, e.g., acacia sodium alginate, polyvinylpyrrolidone, cellulosic polymers (including hydroxypropyl cellulose, hydroxypropyl methylcellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, and the like), and Veegum. Diluents are typically necessary to increase bulk so that a practical size tablet is ultimately provided. Suitable diluents include dicalcium phosphate, calcium sulfate, lactose, cellulose, kaolin, mannitol, sodium chloride, dry starch and powdered sugar. Lubricants are used to facilitate tablet manufacture; examples of suitable lubricants include, for example, vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and oil of theobroma, glycerin, magnesium stearate, calcium stearate, and stearic acid. Stearates, if present, preferably represent at no more than approximately 2 wt. %> of the drug-containing core. Disintegrants are used to facilitate disintegration of the tablet, and are generally starches, clays, celluloses, algins, gums or crosslinked polymers. Fillers include, for example, materials such as silicon dioxide, titanium dioxide, alumina, talc, kaolin, powdered cellulose and microcrystalline cellulose, as well as soluble materials such as mannitol, urea, sucrose, lactose, dextrose, sodium chloride and sorbitol. Stabilizers are used to inhibit or retard drug decomposition reactions that include, by way of example, oxidative reactions. Surfactants may be anionic, cationic, amphoteric or nonionic surface active agents. The pharmaceutical composition comprising FGF-2 and/or FGF-20 or variant thereof can be formulated in solid-dosage form with a permeation-enhancing mixture of sodium salicylate and an oil to provide enhanced absorption of these polypeptides through the wall of the gastrointestinal tract when administered orally. See, for example, U.S. Patent Nos. 5,424,298 and 6,008,187. The dosage form may also be a capsule, in which case the active agent- containing composition may be encapsulated in the form of a liquid or solid

(including particulates such as granules, beads, powders or pellets). Suitable capsules may be either hard or soft, and are generally made of gelatin, starch, or a cellulosic material, with gelatin capsules preferred. Two-piece hard gelatin capsules are preferably sealed, such as with gelatin bands or the like. (See, for e.g., Remington: The Science and Practice of Pharmacy (2000), supra), which describes materials and methods for preparing encapsulated pharmaceuticals. If the FGF-2/FGF-20- containing composition is present within the capsule in liquid form, a liquid carrier is necessary to dissolve the FGF-2 and/or FGF-20. The carrier must be compatible with the capsule material and all components of the pharmaceutical composition, and must be suitable for ingestion.

Solid dosage forms, whether tablets, capsules, caplets, or particulates, may, if desired, be coated so as to provide for delayed release. Dosage forms with delayed release coatings may be manufactured using standard coating procedures and equipment. Such procedures are known to those skilled in the art and described in the pertinent texts (See, for e.g., Remington: The Science and Practice of Pharmacy (2000), supra). Generally, after preparation of the solid dosage form, a delayed release coating composition is applied using a coating pan, an airless spray technique, fluidized bed coating equipment, or the like. Delayed release coating compositions comprise a polymeric material, e.g., cellulose butyrate phthalate, cellulose hydrogen phthalate, cellulose proprionate phthalate, polyvinyl acetate phthalate, cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate, dioxypropyl methylcellulose succinate, carboxymethyl ethylcellulose, hydroxypropyl methylcellulose acetate succinate, polymers and copolymers formed from acrylic acid, methacrylic acid, and/or esters thereof.

Sustained-release dosage forms provide for drug release over an extended time period, and may or may not be delayed release. Generally, as will be appreciated by those of ordinary skill in the art, sustained-release dosage forms are formulated by dispersing a drug within a matrix of a gradually bioerodible (hydrolyzable) material such as an insoluble plastic, a hydrophilic polymer, or a fatty compound, or by coating a solid, drug-containing dosage form with such a material. Insoluble plastic matrices may be comprised of, for example, polyvinyl chloride or polyethylene. Hydrophilic polymers useful for providing a sustained release coating or matrix cellulosic polymers include, without limitation: cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropylmethyl cellulose phthalate, hydroxypropylcellulose phthalate, cellulose hexahydrophthalate, cellulose acetate hexahydrophthalate, and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, acrylic acid alkyl esters, methacrylic acid alkyl esters, and the like, e.g. copolymers of acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and/or ethyl methacrylate, with a terpolymer of ethyl acrylate, methyl methacrylate and trimethylammonioethyl methacrylate chloride (sold under the tradename Eudragit RS) preferred; vinyl polymers and copolymers such as polyvinyl pyrrolidone, polyvinyl acetate, polyvinylacetate phthalate, vinylacetate crotonic acid copolymer, and ethylene- vinyl acetate copolymers; zein; and shellac, ammoniated shellac, shellac- acetyl alcohol, and shellac n-butyl stearate. Fatty compounds for use as a sustained release matrix material include, but are not limited to, waxes generally (e.g., carnauba wax) and glyceryl tristearate.

Topical Administration Topical formulations may be in any form suitable for application to the body surface, and may comprise, for example, an ointment, cream, gel, lotion, solution, paste or the like, and/or may be prepared so as to contain liposomes, micelles, and/or microspheres. Preferred topical formulations herein are ointments, creams, and gels. Furthermore, topical administration of the pharmaceutical composition directly to an extranasal (i.e., outside of the nasal cavity) tissue that is innervated by the trigeminal nerve provides for delivery of the administered FGF-2 and/or FGF-20 to the CNS in a manner that bypasses the obstacle of entry presented by the blood-brain barrier. Ointments, as is well known in the art of pharmaceutical formulation, are semisolid preparations that are typically based on petrolatum or other petroleum derivatives. The specific ointment base to be used, as will be appreciated by those skilled in the art, is one that will provide for optimum drug delivery, and, preferably, will provide for other desired characteristics as well, e.g., emolliency or the like. As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating and nonsensitizing. As explained in Remington: The Science and Practice of Pharmacy (2000), supra, ointment bases may be grouped in four classes: oleaginous bases; emulsifiable bases; emulsion bases; and water-soluble bases. Oleaginous ointment bases include, for example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum. Emulsifiable ointment bases, also known as absorbent ointment bases, contain little or no water and include, for example, hydroxystearin sulfate, anhydrous lanolin and hydrophilic petrolatum. Emulsion ointment bases are either water-in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, and include, for example, cetyl alcohol, glyceryl monostearate, lanolin and stearic acid. Preferred water-soluble ointment bases are prepared from polyethylene glycols of varying molecular weight (See, e.g., Remington: The Science and Practice of Pharmacy (2002), supra).

Creams, as also well known in the art, are viscous liquids or semisolid emulsions, either oil-in-water or water-in-oil. Cream bases are water-washable, and contain an oil phase, an emulsifier and an aqueous phase. The oil phase, also called the "internal" phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol. The aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant. As will be appreciated by those working in the field of pharmaceutical formulation, gels-are semisolid, suspension-type systems. Single-phase gels contain organic macromolecules distributed substantially uniformly throughout the carrier liquid, which is typically aqueous, but also, preferably, contain an alcohol and, optionally, an oil. Preferred "organic macromolecules," i.e., gelling agents, are crosslinked acrylic acid polymers such as the "carbomer" family of polymers, e.g., carboxypolyalkylenes that may be obtained commercially under the Carbopol® trademark. Also preferred are hydrophilic polymers such as polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers and polyvinylalcohol; cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methylcellulose; gums such as tragacanth and xanthan gum; sodium alginate; and gelatin. In order to prepare a uniform gel, dispersing agents such as alcohol or glycerin can be added, or the gelling agent can be dispersed by trituration, mechanical mixing, and/or stirring. Various additives, known to those skilled in the art, may be included in the topical formulations. For example, solubilizers may be used to solubilize certain active agents. For those drugs having an unusually low rate of permeation through the skin or mucosal tissue, it may be desirable to include a permeation enhancer in the formulation; suitable enhancers are as described elsewhere herein.

Intrathecal and Intracerebral Administration

Intrathecal administration, if used, is generally characterized by administration directly into the intrathecal space (where fluid flows around the spinal cord). One common system utilized for intrathecal administration is the APT Intrathecal treatment system available from Medtronic, Inc. APT Intrathecal uses a small pump that is surgically placed under the skin of the abdomen to deliver medication directly into the intrathecal space. The medication is delivered through a small tube called a catheter that is also surgically placed. The medication can then be administered directly to cells in the spinal cord involved in conveying sensory and motor signals. Another system available from Medtronic that is commonly utilized for intrathecal administration is the fully implantable, programmable SynchroMed^® Infusion System. The SynchroMed^® Infusion System has two parts that are both placed in the body during a surgical procedure: the catheter and the pump. The catheter is a small, soft tube. One end is connected to the catheter port of the pump, and the other end is placed in the intrathecal space. The pump is a round metal device about one inch (2.5 cm) thick, three inches (8.5 cm) in diameter, and weighs about six ounces (205 g) that stores and releases prescribed amounts of medication directly into the intrathecal space. It is made of titanium, a lightweight, medical-grade metal. The reservoir is the space inside the pump that holds the medication. The fill port is a raised center portion of the pump through which the pump is refilled. The doctor or a nurse inserts a needle through the patient's skin and through the fill port to fill the pump. Some pumps have a side catheter access port that allows the doctor to inject other medications or sterile solutions directly into the catheter, bypassing the pump.

The SynchroMed^® pump automatically delivers a controlled amount of medication through the catheter to the intrathecal space around the spinal cord, where it is most effective. The exact dosage, rate and timing prescribed by the doctor are entered in the pump using a programmer, an external computer-like device that controls the pump's memory. Information about the patient's prescription is stored in the pump's memory. The doctor can easily review this information by using the programmer. The programmer communicates with the pump by radio signals that allow the doctor to tell how the pump is operating at any given time. The doctor also can use the programmer to change your medication dosage.

Methods of intrathecal administration may include those described above available from Medtronic, as well as other methods that are known to one of skill in the art.

Intracerebral administration can be provided by phleboclysis, endoscopic injection administration, or intracerebral direct injection. Intracerebral administration can also be accomplished by implanting a dosage of the treatment composition(s) incorporated in a non-reactive carrier to provide controlled diffusion of the FGF-2 and/or FGF-20 or variants thereof over a time course to a circumscribed region of the brain, or by perfusion via a mechanized delivery system, such as an osmotic pump.

Intranasal Administration

In addition to the therapeutically effective dose of FGF-2 and or FGF-20 or variants thereof, the pharmaceutical composition for intranasal administration can include, for example, any pharmaceutically acceptable additive, carrier, and/or adjuvant that can promote the transfer of these agents within or through a nasal tissue innervated by the trigeminal nerve or olfactory nerve or along or through a neural pathway. Alternatively, the composition can comprise FGF-2 and/or FGF-20 or variants thereof combined with substances that assist in transporting FGF-2 and FGF- 20 or variants thereof to a transplanted donor cell or other population of CNS cells involved in the progression of a CNS disorder. The composition can further comprise other compounds or components in addition to FGF-2 and FGF-20 or variants thereof so long as the therapeutic efficacy of the FGF-2 or a variant thereof and FGF-20 or a variant thereof is not lessened.

By "pharmaceutically acceptable carrier" is intended a carrier that is conventionally used in the art to facilitate the storage, administration, and/or the biological activity of FGF-2 and FGF-20 or variants thereof. A carrier may also reduce any undesirable side effects of the FGF-2 and FGF-20 or variants thereof. A suitable carrier should be stable, i.e., incapable of reacting with other ingredients in the formulation. It should not produce significant local or systemic adverse effect in recipients at the dosages and concentrations employed for treatment. Such carriers are generally known in the art.

Suitable carriers for an intranasally administered pharmaceutical formation include those conventionally used for large stable macromolecules such as albumin, gelatin, collagen, polysaccharide, monosaccharides, polyvinylpyrrolidone, polylactic acid, polyglycolic acid, polymeric amino acids, fixed oils, ethyl oleate, liposomes, glucose, sucrose, lactose, mannose, dextrose, dextran, cellulose, mannitol, sorbitol, polyethylene glycol (PEG), and the like. Water, saline, aqueous dextrose, and glycols are preferred liquid carriers, particularly (when isotonic) for solutions. The carrier can be selected from various oils, including those of petroleum, animal, vegetable or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Suitable pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. The compositions can be subjected to conventional pharmaceutical expedients, such as sterilization, and can contain conventional pharmaceutical additives, such as preservatives, stabilizing agents, wetting, or emulsifying agents, salts for adjusting osmotic pressure, buffers, and the like.

Other acceptable components in the composition include, but are not limited to, isotonicity-modifying agents such as water, saline, and buffers including phosphate, citrate, succinate, acetic acid, and other organic acids or their salts. Typically, the pharmaceutically acceptable carrier also includes one or more stabilizers, reducing agents, anti-oxidants and/or anti-oxidant chelating agents. The use of buffers, stabilizers, reducing agents, anti-oxidants and chelating agents in the preparation of protein-based compositions, particularly pharmaceutical compositions, is well known in the art. See, Wang et al. (1980) J. Parent. Drug Assn. 34(6):452- 462; Wang et al. (1988) J. Parent. Sci. Tech. 42:S4-S26 (Supplement); Lachman et al. (1968) Drug and Cosmetic Industry 102(l):36-38, 40, and 146-148; Akers (1988) J. Parent. Sci. Tech. 36(5):222-228; and Methods in Enzymology, Vol. XXV, ed. Colowick and Kaplan, "Reduction ofDisulfide Bonds in Proteins with Dithiothreitol," by Konigsberg, pp. 185-188.

Suitable buffers include acetate, adipate, benzoate, citrate, lactate, maleate, phosphate, tartarate, borate, tri(hydroxymethyl aminomethane), succinate, glycine, histidine, the salts of various amino acids, or the like, or combinations thereof. See Wang (1980) supra at page 455. Suitable salts and isotonicifiers include sodium chloride, dextrose, mannitol, sucrose, trehalose, or the like. Where the carrier is a liquid, it is preferred that the carrier is hypotonic or isotonicwith nasal tissue fluids and has a pH within the range of 4.5-8.5. Where the carrier is in powdered form, it is preferred that the carrier is also within an acceptable non-toxic pH range.

Suitable reducing agents, which maintain the reduction of reduced cysteines, include dithiothreitol (DTT also known as Cleland's reagent) or dithioerythritol at 0.01% to 0.1% wt/wt; acetylcysteine or cysteine at 0.1% to 0.5% (pH 2-3); and thioglycerol at 0.1% to 0.5% (pH 3.5 to 7.0) and glutathione. See Akers (1988) supra at pages 225-226. Suitable antioxidants include sodium bisulfite, sodium sulfrte, sodium metabisulfite, sodium thiosulfate, sodium formaldehyde sulfoxylate, and ascorbic acid. See Akers (1988) supra at page 225. Suitable chelating agents, which chelate trace metals to prevent the trace metal catalyzed oxidation of reduced cysteines, include citrate, tartarate, ethylenediaminetetraacetic acid (EDTA) in its disodium, tetrasodium, and calcium disodium salts, and diethylenetriamine pentaacetic acid (DTP A). See, e.g., Wang (1980) supra at pages 457-458 and 460- 461, and Akers (1988) supra at pages 224-227.

The composition can include one or more preservatives such as phenol, cresol, paraaminobenzoic acid, BDSA, sorbitrate, chlorhexidine, benzalkonium chloride, or the like. Suitable stabilizers include carbohydrates such as trehalose or glycerol. The composition can include a stabilizer such as one or more of microcrystalline cellulose, magnesium stearate, mannitol, or sucrose to stabilize, for example, the physical form of the composition; and one or more of glycine, arginine, hydrolyzed collagen, or protease inhibitors to stabilize, for example, the chemical structure of the composition. Suitable suspending agents include carboxymethyl cellulose, hydroxypropyl methylcellulose, hyaluronic acid, alginate, chondroitin sulfate, dextran, maltodextrin, dextran sulfate, or the like. The composition can include an emulsifier such as polysorbate 20, polysorbate 80, pluronic, triolein, soybean oil, lecithins, squalene and squalanes, sorbitan treioleate, or the like. The composition can include an antimicrobial such as phenylethyl alcohol, phenol, cresol, benzalkonium chloride, phenoxyethanol, chlorhexidine, thimerosol, or the like. Suitable thickeners include natural polysaccharides such as mannans, arabinans, alginate, hyaluronic acid, dextrose, or the like; and synthetic ones like the PEG hydrogels of low molecular weight; and aforementioned suspending agents. The composition can include an adjuvant such as cetyl trimethyl ammonium bromide, BDSA, cholate, deoxycholate, polysorbate 20 and 80, fusidic acid, or the like. Suitable sugars include glycerol, threose, glucose, galactose, mannitol, and sorbitol.

Preferred compositions include one or more of a solubility enhancing additive, preferably a cyclodextrin; a hydrophilic additive, preferably a monosaccharide or oligosaccharide; an absorption promoting additive, preferably a cholate, a deoxycholate, a fusidic acid, or a chitosan; a cationic surfactant, preferably a cetyl trimethyl ammonium bromide; a viscosity enhancing additive, preferably to promote residence time of the composition at the site of administration, preferably a carboxymethyl cellulose, a maltodextrin, an alginic acid, a hyaluronic acid, or a chondroitin sulfate; or a sustained release matrix, preferably a polyanhydride, a polyorthoester, a hydrogel, a particulate slow release depo system, preferably a polylactide co-glycolides (PLG), or a starch microsphere; a lipid-based carrier, preferably an emulsion, a liposome, a niosome, or a micelle. The composition can include a bilayer destabilizing additive, preferably a phosphatidyl ethanolamine; a fusogenic additive, preferably a cholesterol hemisuccinate.

These lists of carriers and additives are by no means complete, and a worker skilled in the art can choose excipients from the GRAS (generally regarded as safe) list of chemicals allowed in the pharmaceutical preparations and those that are currently allowed in topical and parenteral formulations.

The method for formulating a pharmaceutical composition is generally known in the art. A thorough discussion of formulation and selection of pharmaceutically acceptable carriers, stabilizers, and isomolytes can be found in Remington: The Science and Practice of Pharmacy (2000), supra, herein incorporated by reference.

Among the optional substances that may be combined with the FGF-2 and FGF-20 or variants thereof in the pharmaceutical composition are lipophilic substances that can enhance absorption of the FGF-2 and FGF-20 or variants thereof through the mucosa or epithelium of the nasal cavity to damaged cells in the CNS. The FGF-2 and FGF-20 or variants thereof may be mixed with a lipophilic adjuvant alone or in combination with a carrier, or may be combined with one or several types of micelle or liposome substances. Among the preferred lipophilic substances are cationic liposomes including one or more of phosphatidyl choline, lipofectin, DOTAP, or the like. These liposomes may include other lipophilic substances such as gangliosides and phosphatidylserine (PS). Also preferred are micellar additives such as GM-1 gangliosides and phosphatidylserine (PS), which may be combined with the FGF-2 and FGF-20 or variants thereof either alone or in combination. GM-1 ganglioside can be included at 1-10 mole percent in any liposomal compositions or in higher amounts in micellar structures. Protein agents can be either encapsulated in particulate structures or incorporated as part of the hydrophobic portion of the structure depending on the hydrophobicity of the protein agent. One preferred liposomal formulation employs Depofoam. The neuroprotective agent can be encapsulated in multivesicular liposomes, as disclosed in the copending application entitled "High and Low Load Formulations ofIGF-I in Multivesicular Liposomes," International Publication No. WO 99/12522, herein incorporated by reference. In one embodiment, the composition includes the combination of an effective amount of growth factor with poly(ethylene-co-vinyl acetate) to provide for controlled release of these growth factors. A composition formulated for intranasal delivery may optionally comprise an odorant. An odorant agent is combined with the FGF-2 and FGF-20 or variants thereof to provide an odoriferous sensation, and/or to encourage inhalation of the intranasal preparation to enhance delivery of the FGF-2 and FGF-20 or variants thereof to the olfactory neuroepithelium. The odoriferous sensation provided by the odorant agent may be pleasant, obnoxious, or otherwise malodorous. The odorant receptor neurons are localized to the olfactory epithelium, which, in humans, occupies only a few square centimeters in the upper part of the nasal cavity. The cilia of the olfactory neuronal dendrites which contain the receptors are fairly long (about 30-200 um). A 10-30 μm layer of mucus envelops the cilia that the odorant agent must penetrate to reach the receptors. See Snyder et al. (1988) J Biol. Chem. 263: 13972- 13974. Use of a lipophilic odorant agent having moderate to high affinity for odorant binding protein (OBP) is preferred. OBP has an affinity for small lipophilic molecules found in nasal secretions and may act as a carrier to enhance the transport of a lipophilic odorant substance and active FGF-2 and FGF-20 or variants thereof to the olfactory receptor neurons. It is also preferred that an odorant agent is capable of associating with lipophilic additives such as liposomes and micelles within the preparation to further enhance delivery of the FGF-2 and FGF-20 or variants thereof by means of OBP to the olfactory neuroepithelium. OBP may also bind directly to lipophilic agents to enhance transport of the FGF-2 and FGF-20 or variants thereof to olfactory neural receptors.

Suitable odorants having a high affinity for OBP include terpanoids such as cetralva and citronellol, aldehydes such as amyl cirmamaldehyde and hexyl cirmamaldehyde, esters such as octyl isovalerate, jasmines such as Cl S-jasmine and jasmal, and musk 89. Other suitable odorant agents include those which may be capable of stimulating odorant-sensitive enzymes such as adenylate cyslase and guanylate cyclase, or which may be capable of modifying ion channels within the olfactory system to enhance absorption of the FGF-2 and FGF-20 or variants thereof. For the purposes of this invention, the pharmaceutical composition comprising the FGF-2 and/or FGF-20 or variants thereof can be formulated in a unit dosage and in a form such as a solution, suspension, or emulsion. The composition can also be in the form of lyophilized powder, which can be converted into solution, suspension, or emulsion before intranasal administration. The pharmaceutical composition comprising the FGF-2 and or FGF-20 or variants thereof is preferably sterilized by membrane filtration and is stored in unit-dose or multi-dose containers such as sealed vials or ampoules. Preferably, the volume of one dose of the pharmaceutical composition ranges from about 10 μl to about 0.2 ml, preferably from about 50 μl to about 200 μl for each of the FGF-2 and FGF-20, or variants thereof. Alternatively, the volume of one dose may be about 10 μl, 50 μl, 100 μl, 150 μl, or 200 μl for each of the FGF-2 and FGF-20, or variants thereof. It is apparent that the suitable volume can vary with factors such as the size of the nasal cavity to which the FGF-2 and/or FGF-20 or variants thereof are administered and the solubility of the components in the composition.

Dosages

The concentration of FGF-2 and/or FGF-20 in any of the aforementioned dosage forms and compositions can vary a great deal, and will depend on a variety of factors, including the type of composition or dosage form, the corresponding mode of administration, the nature and activity of the specific active agent, and the intended drug release profile.

Preferred dosage forms contain a unit dose of these therapeutic agents, i.e., a single therapeutically effective dose. For creams, ointments, etc., a "unit dose" requires an active agent concentration that provides a unit dose in a specified quantity of the formulation to be applied. The unit dose of any particular active agent will depend, of course, on the active agent and on the mode of administration. For FGF-2 and FGF-20, and variants thereof, the unit dose for oral administration of each of these therapeutic agents will be in the range of from about 1 mg to about 10,000 mg, typically in the range of from about 100 mg to about 5,000 mg. Alternatively, for FGF-2 and FGF-20, and variants thereof, the unit dose for oral administration of each of these therapeutic agents will be greater than about 1 mg, about 5 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about

1,000 mg, about 1,500 mg, about 2,000 mg, about 2,500 mg, about 3,000 mg, about 3,500 mg, about 4,000 mg, about 4,500 mg, about 5,000 mg, about 5,500 mg, about 6,000 mg, about 6,500 mg, about 7,000 mg, about 7,500 mg, about 8,000 mg, about 8,500 mg, about 9,000 mg, or about 9,500 mg. Those of ordinary skill in the art of pharmaceutical formulation can readily deduce suitable unit doses for FGF-2 and FGF-20, and variants thereof, as well as suitable unit doses for other types of agents that may be incorporated into a dosage form of the invention. For FGF-2 and FGF-20, and variants thereof, the unit dose for transmucosal, topical, transdermal, and parenteral administration of each of these therapeutic agents will be in the range of from about 1 ng to about 10,000 mg, typically in the range of from about 100 ng to about 5,000 mg. Alternatively, for FGF-2 and FGF-20, and variants thereof, the unit dose for transmucosal, topical, transdermal, and parenteral administration for each of these therapeutic agents will be greater than about 1 ng, about 5 ng, about 10 ng, about 20 ng, about 30 ng, about 40 ng, about 50 ng, about 100 ng, about 200 ng, about 300 ng, about 400 ng, about 500 ng, about 1 μg, about 5 μg, about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 100 μg, about 200 μg, about 300 μg, about 400 μg, about 500 μg, about 1 mg, about 5 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 1,000 mg, about 1,500 mg, about 2,000 mg, about 2,500 mg, about 3,000 mg, about 3,500 mg, about 4,000 mg, about 4,500 mg, about 5,000 mg, about 5,500 mg, about 6,000 mg, about 6,500 mg, about 7,000 mg, about 7,500 mg, about 8,000 mg, about 8,500 mg, about 9,000 mg, or about 9,500 mg. Those of ordinary skill in the art of pharmaceutical formulation can readily deduce suitable unit doses for FGF-2 and FGF-20, and variants thereof, as well as suitable unit doses for other types of agents that may be incorporated into a dosage form of the invention. For FGF-2 and FGF-20, and variants thereof, the unit dose for intrathecal administration for each of these therapeutic agents will be in the range of from about 1 fg to about 1 mg, typically in the range of from about 100 fg to about 1 ng. Alternatively, for FGF-2 and FGF-20, and variants thereof, the unit dose for intrathecal administration for each active agent will be greater than about 1 fg, about 5 fg, about 10 fg, about 20 fg, about 30 fg, about 40 fg, about 50 fg, about 100 fg, about 200 fg, about 300 fg, about 400 fg, about 500 fg, about 1 pg, about 5 pg, about 10 pg, about 20 pg, about 30 pg, about 40 pg, about 50 pg, about 100 pg, about 200 pg, about 300 pg, about 400 pg, about 500 pg, about 1 ng, about 5 ng, about 10 ng, about 20 ng, about 30 ng, about 40 ng, about 50 ng, about 100 ng, about 200 ng, about 300 ng, about 400 ng, about 500 ng, about 1 μg, about 5 μg, about 10 μg, about 20 μg, about

30 μg, about 40 μg, about 50 μg, about 100 μg, about 200 μg, about 300 μg, about 400 μg, or about 500 μg. Those of ordinary skill in the art of pharmaceutical formulation can readily deduce suitable unit doses for FGF-2 and FGF-20, and variants thereof, as well as suitable unit doses for other types of agents that may be incorporated into a dosage form of the invention.

A therapeutically effective amount of a particular active agent administered to a given individual will, of course, be dependent on a number of factors, including the concentration of the specific growth factor agent, composition or dosage form, the selected mode of administration, the age and general condition of the individual being treated, the severity of the individual's condition, and other factors known to the prescribing physician.

Articles and Methods of Manufacture

The present invention also includes an article of manufacture providing FGF-2 and FGF-20, or variants thereof, for administration to the CNS. The article of manufacture can include a vial or other container that contains a composition suitable for the present method together with any carrier, either dried or in liquid form. The article of manufacture further includes instructions in the form of a label on the container and/or in the form of an insert included in a box in which the container is packaged, for the carrying out the method of the invention. The instructions can also be printed on the box in which the vial is packaged. The instructions contain information such as sufficient dosage and administration information so as to allow the subject or a worker in the field to administer the FGF-2 and FGF-20 or a variant thereof. It is anticipated that a worker in the field encompasses any doctor, nurse, technician, spouse, or other care-giver that might administer the FGF-2 and FGF-20 or a variant thereof. The FGF-2 and FGF-20, or variants thereof, can also be self-administered by the subject. According to the invention, FGF-2 and FGF-20, or variants thereof can be used for manufacturing FGF-2 and FGF-20, or variants thereof, as a composition or medicament suitable for administration by any acceptable route, for example, for parenteral, transdermal, transmucosal, inhalation, or nasal administration. For example, a liquid or solid composition can be manufactured in several ways, using conventional techniques. A liquid composition can be manufactured by dissolving FGF-2 and FGF-20, or variants thereof, in a suitable solvent, such as water, at an appropriate pH, including buffers or other excipients, for example to form a solution described herein above. Biologically Active Variants of FGF-2 and FGF-20

As noted above, the invention has been described with reference to FGF-2 and FGF-20, though biologically active variants as defined below can also be used in the methods presented herein and in formulating the pharmaceutical compositions for practicing methods of treatment disclosed herein. Variants of an FGF sequence include, but are not limited to, biologically active fragments, analogues, and derivatives.

By "fragment" is intended a polypeptide consisting of only a part of the intact FGF-2 or FGF-20 sequence and structure, and can be a C-terminal deletion, N- terminal deletion, or both. A fragment of a native-sequence FGF-2 (for example, human FGF-2 of SEQ ID NO:2) or a native-sequence FGF-20 (for example, human FGF-20 of SEQ ID NO:4) can comprise 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 amino acids or up to the full length of the native sequence FGF-2 or FGF-20 molecule (for example, up to 146 residues for the 146-residue form of human FGF-2 of SEQ ID NO:2, or up to 155 residues for native human FGF-2 of SEQ ID NO:6; or up to 211 residues for human FGF-20 of SEQ ID NO: 10). Similarly, with respect to coding sequences, fragments of a nucleotide sequence encoding a functional fragment of human FGF-2 may range from at least 45, 60, 75, 90, 105, 120, 135, 150, 165, 180, 195, 210, 225, 240, 255, 270, 285, 300, 315, 330, 345, 360, 375, 390, 405, 420, 435, 450, 460 nucleotides, and up to the entire length of the nucleotide sequence encoding the native-sequence FGF-2 or FGF-20 polypeptide (for example, up to the full-length nucleotide sequence set forth in SEQ ID NO:l, which encodes human FGF-2 of SEQ ED NO:2, or up to the full-length nucleotide sequence set forth in SEQ ID NO: 9, which encodes human FGF-20 of SEQ ID NO: 10).

By "analogues" is intended analogues of either the FGF-2 or FGF-20 or fragments thereof that comprise a native FGF-2 or FGF-20 sequence and structure having one or more amino acid substitutions, insertions, or deletions. Peptides having one or more peptoids (peptide mimics) and muteins, or mutated forms of the FGF-2 or FGF-20, are also encompassed by the term analogue. By "derivatives" is intended any suitable modification of the FGF-2 or FGF-20, fragments of the FGF-2 or FGF- 20, or their respective analogues, such as glycosylation, phosphorylation, or other addition of foreign moieties, so long as the angiogenic activity is retained. Methods for making fragments, analogues, and derivatives are available in the art. See generally U.S. Patent Nos. 4,738,921, 5,158,875, and 5,077,276; International Publication Nos. WO 85/0083 1, WO 92/04363, WO 87/01038, and WO 89/05822; and European Patent Nos. EP 135094, EP 123228, and EP 128733; herein incorporated by reference.

By "sequence identity" is intended the same nucleotides or amino acid residues are found within the variant sequence and a reference sequence when a specified, contiguous segment of the nucleotide sequence or amino acid sequence of the variant is aligned and compared to the nucleotide sequence or amino acid sequence of the reference sequence. Methods for sequence alignment and for determining identity between sequences are well known in the art. See, for example, Ausubel et al, eds. (1995) Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978) in Atlas of Polypeptide Sequence and Structure 5:Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.). With respect to optimal alignment of two nucleotide sequences, the contiguous segment of the variant nucleotide sequence may have additional nucleotides or deleted nucleotides with respect to the reference nucleotide sequence. Likewise, for purposes of optimal alignment of two amino acid sequences, the contiguous segment of the variant amino acid sequence may have additional amino acid residues or deleted amino acid residues with respect to the reference amino acid sequence. The contiguous segment used for comparison to the reference nucleotide sequence or reference amino acid sequence will comprise at least 20 contiguous nucleotides, or amino acid residues, and may be 30, 40, 50, 100, or more nucleotides or amino acid residues. Corrections for increased sequence identity associated with inclusion of gaps in the variant's nucleotide sequence or amino acid sequence can be made by assigning gap penalties. Methods of sequence alignment are well known in the art.

For purposes of the present invention, percent sequence identity at the amino acid level is determined using the Smith- Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2,

BLOSUM matrix of 62. The Smith- Waterman homology search algorithm is taught in Smith and Waterman, Adv. Appl. Math. (1981) 2:482-489. Alternatively, percent identity of a nucleotide sequence is determined using the Smith- Waterman homology search algorithm using a gap open penalty of 25 and a gap extension penalty of 5. Such a determination of sequence identity can be performed using, for example, the DeCypher Hardware Accelerator from TimeLogic. It is further recognized that when considering percentage of amino acid identity, some amino acid positions may differ as a result of conservative amino acid substitutions, which do not affect properties of polynucleotide function. In these instances, percent sequence identity may be adjusted upwards to account for the similarity in conservatively substituted amino acids. Such adjustments are well known in the art. See, for example, Meyers et al. (1988) Computer Applic. Biol. Sci. 4:11-17.

For example, amino acid sequence variants of native-sequence human FGF-2 or native-sequence human FGF-20 can be prepared by mutations in the respective cloned DNA sequences encoding these polypeptides, which are set forth in SEQ ID NO: 1 and SEQ ID NO: 9, respectively, or by mutations in a nucleotide sequence that encodes human FGF-2 or human FGF-20 but which differs from SEQ ID NO:l or SEQ ID NO:9, respectively, due to degeneracy of the genetic code. Such variant nucleotide sequences can be naturally occurring allelic variants, such as those identified with the use of well-known molecular biology techniques, such as polymerase chain reaction and hybridization techniques. Naturally occurring allelic variations can typically result in l-5%> variance in the nucleotide sequence of the native gene. Variant nucleotide sequences encoding human FGF-2 or human FGF-20 can also be synthetically derived nucleotide sequences generated, for example, by site-directed mutagenesis of a naturally occurring nucleotide sequence. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods Enzymol. 154:367-382; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Plainview, New York); U.S. Patent No. 4,873,192; and the references cited therein; herein incorporated by reference. Generally, nucleotide sequence variants for use in preparing polypeptide variants of a native-sequence FGF-2, for example human

FGF-2, or native-sequence FGF-20, for example human FGF-20, will have at least 70%, generally 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to a native coding sequence for FGF-2, for example, the sequence set forth in SEQ ED NO:l (encoding native human FGF-2) or the sequence set forth in SEQ ID NO: 9 (encoding native human FGF-20), as determined using the sequence alignment program identified herein above.

Guidance as to appropriate amino acid substitutions that do not affect biological activity of a given polypeptide may be found in the model of Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure (National Biomedical Research Foundation, Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferred. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). See, for example, Bowie et al. (1990) Science 247:1306, herein incorporated by reference. By "charged amino acids" is intended those amino acids with either a cationic (Lys, Arg, His) or anionic (Asp, Glu) charge. Examples of conservative substitutions include, but are not limited to, Gly<=>Ala, Val<=>Ile<=>Leu, Asp = Glu, Lys«Arg, Asn = Gln, and Phe<=>Trp<»Tyr. Examples of nonconservative amino acids include, but are not limited to, Ala<=>Thr, AspoGly, and Ala<= Ser. In constructing variants of human FGF-20, modifications are made such that variants continue to possess the desired activity, i.e., enhancing neuronal survival. Obviously, any mutations made in the DNA encoding the variant polypeptide must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See EP Patent Application Publication No. 75,444. Biologically active variants of a native-sequence FGF-2 or native-sequence FGF-20 polypeptide will generally have at least 70%, 75%, 80%, generally at least 85%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and more preferably at least 98% or even at least 99% or more amino acid sequence identity to the amino acid sequence of the reference FGF-2 polypeptide (for example, SEQ ID NO:2 for human FGF-2) or the reference FGF-20 polypeptide (for example, SEQ ID NO: 10 for human FGF-20), which serves as the basis for comparison. A biologically active variant of a native sequence FGF-2 or native sequence FGF-20 polypeptide may differ from the respective native polypeptide by as few as 1-15 amino acids, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue. In constructing a variant of a native FGF-2 or native FGF-20 polypeptide, any deletions, insertions, and/or substitutions to the sequence encoding the native polypeptide are not expected to produce radical changes in the characteristics of the particular variant polypeptide. Thus, variants of an FGF-2 or FGF-20 polypeptide, for example, human FGF-2 or human FGF-20, should retain the desired biological activity of the native sequence, for example, native human FGF-2 or native human FGF-20. Methods are available in the art for determining whether a variant polypeptide retains the desired biological activity of the native polypeptide. Biological activity can be measured using assays specifically designed for measuring activity of the native polypeptide or protein, including assays described in the present invention. Additionally, antibodies raised against the native sequence, for example native human FGF-2 or native human FGF-20, polypeptide can be tested for their ability to bind to the variant FGF-2 or variant FGF-20 polypeptide, respectively, where effective binding is indicative of a polypeptide having a conformation similar to that of the native FGF polypeptide.

For purposes of the present invention, the FGF-20 biological activity of interest is neurotrophic activity for cultured neurons, for example, cultured midbrain dopaminergic neurons. Assays to determine neurotrophic activity of FGF-20 are well known in the art. See, for example, International Publication No. WO 01/31008, herein incoφorated by reference in its entirety. Thus, a biologically active variant of native-sequence human FGF-20 retains the biological activity of native-sequence human FGF-20, that is, the ability to enhance survival of neurons, particularly midbrain dopaminergic neurons, when cultured in medium in the presence of the variant FGF-20 polypeptide relative to survival of these neurons when cultured in the same medium in the absence of the variant FGF-20 polypeptide. Though the variant FGF-20 polypeptide retains the ability to enhance neuronal survival, its level of potency is not necessarily the same as the potency of native human FGF-20 or the potency of recombinantly produced human FGF-20.

For puφoses of the present invention, the FGF-2 biological activity of interest is synergistic promotion of growth, proliferation, differentiation, and/or survival of a CNS cell, particularly dopaminergic neurons, when administered in combination with FGF-20. Assays to determine such biological activity are disclosed elsewhere herein; see the Examples disclosed below. Thus, a biologically active variant of native- sequence human FGF-2 retains the biological activity of native-sequence human FGF- 2, that is, in the presence of FGF-20 or a variant thereof, the ability to synergistically promote growth, proliferation, differentiation, and/or survival of a CNS cell, particularly dopaminergic neurons, when cultured in medium in the presence of a combination of the variant FGF-2 polypeptide and FGF-20 or a variant thereof relative to growth, proliferation, differentiation, and/or survival of these cells when cultured in the same medium with FGF-20 or variant thereof in the absence of the variant FGF-2 polypeptide. Though the variant FGF-2 polypeptide retains the ability to promote growth, proliferation, differentiation, and/or survival of a CNS cell in the presence of FGF-20 or a variant thereof, its level of potency is not necessarily the same as the potency of native human FGF-2 or the potency of recombinantly produced human FGF-2.

The precise chemical structure of a polypeptide having native-sequence FGF-2 or native-sequence FGF-20 biological activity depends on a number of factors. As ionizable amino and carboxyl groups are present in these individual molecules, a particular polypeptide may be obtained as an acidic or basic salt, or in neutral form. All such preparations that retain their biological activity as defined above when placed in suitable environmental conditions are included in the definition of polypeptides having the suitable FGF-2 or FGF-20 biological activity as used herein. Further, the primary amino acid sequence of the polypeptide may be augmented by derivatization using sugar moieties (glycosylation) or by other supplementary molecules such as lipids, phosphate, acetyl groups and the like. It may also be augmented by conjugation with saccharides. Certain aspects of such augmentation are accomplished through post-translational processing systems of the producing host; other such modifications may be introduced in vitro. In any event, such modifications are included in the definition of an FGF-2 or FGF-20 polypeptide used herein so long as the FGF-2 or FGF-20 biological activity of the polypeptide of interest is not destroyed. It is expected that such modifications may quantitatively or qualitatively affect the activity, either by enhancing or diminishing the activity of the polypeptide, in the biological activity assay for neurotrophic activity and promotion of neuronal survival. Further, individual amino acid residues in the chain may be modified by oxidation, reduction, or other derivatization, and the polypeptide may be cleaved to obtain fragments that retain activity. Such alterations that do not destroy activity do not remove the polypeptide sequence from the definition of suitable FGF-2 or FGF-20 polypeptides of interest as used herein.

The art provides substantial guidance regarding the preparation and use of polypeptide variants. In preparing variants of a native-sequence FGF-2 (for example, human FGF-2) or native-sequence FGF-20 (for example, human FGF-20), one of skill in the art can readily determine which modifications to the reference FGF-2 or FGF- 20 nucleotide or amino acid sequence will result in a variant polypeptide that is suitable for use in the methods of treatment described herein, and for use as a therapeutically active component of a pharmaceutical composition described herein. For non-limiting examples of variant FGF-20 polypeptides, see International

Publication No. WO 01/31008. Variants of FGF-2 are also known in the art, including, for example, naturally occurring and biologically active fragments of FGF- 2 that have N-terminal truncations relative to the FGF-2 of SEQ ID NO:4. An active and truncated FGF-2 having residues 12-146 of SEQ ID NO:4 was found in bovine liver and another active and truncated bFGF-2, having residues 16-146 of SEQ ID NO:4 was found in the bovine kidney, adrenal glands, and testes. (See U.S. Patent No. 5,155,214, citing to Ueno et al. (1986) Biochem. Biophys. Res. Comm. 138:580- 588, herein incoφorated by reference.) Likewise, other fragments of the FGF-2 of SEQ ED NO:4 that are known to have FGF activity are FGF-2 (24-120)-OH and FGF-2 (30-110)-NH₂. See U.S. Patent No. 5,155,214, herein incoφorated by reference. These latter fragments retain both of the cell binding portions of FGF-2

(SEQ ED NO:4) and one of the heparin binding segments (residues 107-111).

Accordingly, the biologically active fragments of a mammalian FGF typically encompass those terminally truncated fragments of an FGF-2 that have at least residues that correspond to residues 30-110 of FGF-2 of SEQ ID NO:4; more typically, at least residues that correspond to residues 18-146 of FGF-2 of SEQ ID NO:4. Human and bovine FGF-2 are described in U.S. Patent No. 5,439,818 and U.S. Patent No. 5,155,214, respectively. Several variants (i.e., analogues, derivatives, and fragments) of bFGF are also described in, for example, U.S. Patent No. 5,851,990, Zhu et al. (1991) Science 257:90-93, Biochem. Biophys. Comm. 151 (1988):701-708, EP No. 281,822, EP No. 326,951, EP No. 298,728, EP No. 320,148, EP No. 319,052, EP No. 298,723, EP No. 363,675, WO 89/04832, and U.S. Patent No. 5,310,883; all of which are herein incoφorated by reference.

Recombinantly produced FGF-2 or FGF-20 molecules may be modified further so long as they retain native-sequence FGF-2 or FGF-20 biological activity as noted herein above. Further modifications include, but are not limited to, phosphorylation, substitution of non-natural amino acid analogues, and the like. Modifications to these recombinantly produced FGF-2 or FGF-20 molecules that may lead to prolonged in vivo exposure, and hence increase efficacy of pharmaceutical formulations comprising these recombinantly produced FGF-2 or FGF-20 molecules, include glycosylation or PEGylation of the protein molecule. Glycosylation of proteins not natively glycosylated is usually performed by insertion of N-linked glycosylation sites into the molecule. This approach can be used to prolong half-life of proteins such as recombinant human FGF-2 or recombinant human FGF-20. In addition, this approach can be used to shield immunogenic epitopes, increase protein solubility, reduce aggregation, and increase expression and purification yields. The methods and pharmaceutical compositions of the invention also contemplate the use of FGF-2 and or FGF-20 fusion proteins or polypeptides. A

"fusion protein" or "fusion polypeptide" is a protein or polypeptide resulting from the expression of at least one operably linked heterologous coding sequence. For puφoses of the present invention, the terms "fusion protein" and "fusion polypeptide" are used interchangeably. Routine techniques for the construction of the vectors comprising fusion proteins of a polypeptide such as FGF-2 or FGF-20 are well known to those of ordinary skill in the art and can be found in such references as Sambrook et al. (1989)

Molecular Cloning: A Laboratory Manual (2^nd ed.;Cold Spring Harbor Laboratory Press, Plainview, New York). A variety of strategies are available for ligating fragments of DNA, the choice of which depends on the nature of the termini of the DNA fragments and which choices can be readily made by those of skill in the art. Generally, and unless otherwise specified, the 3' end of the DNA segment encoding the desired rhFGF-20 is ligated in frame to the 5' end of a DNA segment encoding the desired carrier sequence, for example, a signal sequence of a heterologous protein to promote secretion of the protein product, such that a contiguous fusion protein is produced upon expression of the ligated DNA. These strategies may encompass PCR techniques in obtaining or modifying pertinent DNA segments. Also available to one of ordinary skill in the art are a variety of host cells for containing and expressing the desired constructs.

Further, the FGF-2 and/or FGF-20 polypeptide, or biologically active variants thereof, can be constructed as a chimeric peptide to facility peptide drug delivery to the CNS from the bloodstream. Chimeric peptides are formed when a non- transportable peptide therapeutic, such as FGF-2 or FGF-20, is coupled to a blood- brain barrier drug transport vector. Transport vectors include proteins, such as, for example, cationized albumin, or the OX26 monoclonal antibody to the transferring receptor, to allow for absortive-mediated and receptor-mediated transcytosis, respectively, through the blood-brain barrier. Other chimeric peptide strategies include design strategies for coupling drugs to the vector that give high efficiency coupling, thereby resulting in the liberation of biologically active peptides following cleavage of the bond linking the therapeutic and the transport vector. The avidin/biotin system is an example of such a linker-based strategy. See, for example, Bickel et al. (2001) Adv. Drug Deliv. Rev. 46(103):247-279, and Song et al. (2002) J. Pharmacol. Expl. Ther. 301(2):605-610; herein incoφorated by reference in their entirety.

In Vitro Methods for Culturing Neural Progenitor Cells

The present invention also provides a method for promoting differentiation of dopaminergic neurons from a population of neural progenitor cells. The method comprises culturing a population of neural progenitor cells that comprises at least one neural progenitor cell that is capable of differentiating into neurons and glia in a culture medium that provides for differentiation of neural progenitor cells into neurons and glia, and which comprises FGF-2 or variant thereof and FGF-20 or variant thereof. The FGF-2 or variant thereof and FGF-20 or variant thereof are present in the culture medium in amounts effective to promote differentiation of dopaminergic neurons from the neural progenitor cells. Suitable amounts of FGF-2 and FGF-20, or variants thereof, for promoting differentiation of dopaminergic neurons from neural progenitor cells are in the range of about 1 picamole (pM) to about 50 nanomole (nM) of FGF-20 or variant thereof, preferably about 100 pM to about 10 nM of FGF-20 or variant thereof, and about 1 pM to about 50 nM of FGF-2 or biologically active variant thereof, preferably about 50 pM to about 1 nM FGF-2 or biologically active variant thereof. As noted above, in some embodiments, the FGF- 20 or variant there is present in the culture medium in the range from about 100 pM to about 10 nM, including, for example, 100 pM, 150 pM, 200 pM, 250 pM, 300 pM, 350 pM, 400 pM, 450 pM, 500 pM, 550 pM, 600 pM, 650 pM, 700 pM, 750 pM, 800 pM, 850 pM, 900 pM, 950 pM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, and other such values between about 100 pM to about 10 nM, and the FGF-2 or biologically active variant thereof is present in the sutiable culture medium in the range from about 50 pM to about 1 nM, including, for example, 50 pM, 75 pM, 100 pM, 150 pM, 200 pM, 250 pM, 300 pM, 350 pM, 400 pM, 450 pM, 500 pM, 550 pM, 600 pM, 650 pM, 700 pM, 750 pM, 800 pM, 850 pM, 900 pM, 950 pM, 1 nM, and other such values between about 50 pM and about 1 nM. In one embodiment, the suitable culture medium comprises about 1 nM to about 10 nM FGF-20 or biologically active variant thereof, and about 50 pM to about 1 nM of FGF-2 or biologically active variant thereof.

As previously noted, FGF-20 promotes differentiation of dopaminerigc neurons from cultured neural progenitor cells and/or promotes survival of these differentiated dopaminergic neurons during culture, in a concentration-dependent manner (see the Examples disclosed herein below). This effect is maximal in the presence of FGF-2. Thus, a population of neural progenitor cells cultured in a suitable medium that includes the presence of both FGF-2 and FGF-20 as noted herein above comprises a higher percentage of differentiated dopaminergic neurons that does a population of neural progenitor cells cultured in the same medium in the absence of the combination of FGF-2 and FGF-20. As noted above, suitable media for culturing neural progenitor cells are known in the art. See, for example, the media disclosed in Kawasaki et al. (2000) Neuron. 28:31-40 and Kawasaki et al. (2002) Proc. Natl. Acad. Sci. USA 99:1580-1585; herein incoφorated by reference in their entirety.

The resulting population of cultured neural progenitors is enriched in dopaminergic neurons. By "enriched" is intended the population of cultured neural progenitors comprises at least 5%, 8%, 10%, 15%, or 20% of the cells as dopaminergic neurons, preferably at least 25%, 30%, or 35%>, and more preferably at least 40%), 45%, or 50%> of the cells as dopaminergic neurons, depending upon the concentration of FGF-2 and FGF-20, or biologically active variants thereof, present in the culture medium. A composition comprising such a population of cultured neural progenitors is advantageously used in a cell transplantation therapy for a subject suffering from a neurodegenerative disorder, particularly Parkinson's disease, as noted herein above. Detection of increased numbers of dopaminergic neurons in the cultured neural progenitor cells relative to a population of neural progenitor cells cultured in the same culture medium except for the absence of the combination of FGF-2 and FGF-20, or variants thereof, can be accomplished by various assays, including RT-PCR and immunocytochemical methods described elsewhere herein.

The neural progenitor cells can be of any origin, as previously disclosed above. In one embodiment, the subject is a human, and the neural progenitor cells are of human origin. In one such embodiment, the neural progenitor cells are derived from human embryonic stem cells using methods known in the art and disclosed herein above.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

Parkinson's disease (PD) is a degenerative disorder characterized by a loss of midbrain dopaminergic neurons with a subsequent reduction in the level of striatal dopamine (Bergman and Deuschl (2002) Mov. Disord. 17 (Suppl 3):S28-40; Miyasaki and Martin (2002) Neurology 58:1 1-17). Pharmacological treatment with L-DOPA works initially, but reduced efficacy and development of motor complications requires additional treatments such as deep brain stimulation and fetal dopaminergic neurotransplantation (Bergman and Deuschl (2002) Mov. Disord. 17 (Suppl 3):S28- 40; Miyasaki and Martin (2002) Neurology 58:11-17; Dostrovsky et al. (2002) Neuroscientist 8:284-290; Widner et al. (1992) New Engl. J. Med. 32:1556-1563). There is evidence from both animal models and clinical investigations showing that fetal dopaminergic neurons can produce symptomatic relief (Dostrovsky et al. (2002) Neuroscientist 8:284-290; Widner et al. (1992) New Engl. J. Med. 32:1556-1563; Freed et al. (2001) New Engl. J. Med. 344:710-719; Hagell (2001) J. Neuropathol. Exp. Neurol. 60:741-752; Lindvall et al. (1994) Ann. Neurol. 35:172-180).

Embryonic stem (ES) cells have many characteristics required for an optimal cell source for cell-replacement therapy (Smith (2001) Ann. Rev. Cell. Dev. Biol.

17:435-462). ES cells are self-renewing and multipotent cells derived from the inner cell mass of the implantation blastocyst. The presence of a strong neuralization- inducing activity on the cell surface of stromal cells has previously been noted and termed SDIA (stromal cell-derived inducing activity) (Kawasaki et al. (2000) Neuron. 28:31-40). Originally, in the absence of exogenous BMP4, mouse ES cells were shown to differentiate efficiently into neural precursors and neurons when cultured on SDIA-possessing mouse stromal cells (PA6 cells) for 1 week (Kawasaki et al. (2000) Neuron. 28:31-40). Methods for obtaining primate ES cells have been previously established (Thompson et al. (1995) Proc. Natl. Acad. Sci. USA 92:7844-7 48; Suemori et al. (2001) E»ev. Dyn. 222:273-279). Recently, the SDIA method has also become available for primate ES cells. After having been cultured on PA6 cells for 2 weeks, the majority of primate ES colonies contained neural precursors and postmitotic neurons (Kawasaki et al. (2002) Proc. Natl. Acad. Sci. USA 99:1580- 1585). Neural progenitors have multipotent and self-renewal capacities and can be cultured as neurospheres (Gage (2000) Science 287:1433-1438). In the present study, neural progenitors were generated from monkey embryonic stem (ES) cells as neurospheres containing a large number of dopaminergic neurons. The effect of FGF- 20 in combination with FGF-2 on the growth and survival of these dopaminergic neurons in culture was assessed. FGF-20 in combination with FGF-2 could significantly expand the number of these dopaminergic neurons. These dopaminergic neurons were then transplanted into MPTP-treated monkeys as a primate model of

Parkinson's disease. Behavioral studies and functional imaging revealed that the transplanted cells functioned as dopaminergic neurons and could attenuate the MPTP- induced symptoms.

Example 1 : Maintenance of Primate Embryonic Stem Cells Cynomolgus monkey embryonic stem (ES) cell lines were established, and their pluripotency was confirmed by teratoma formation in mice with severe combined immunodeficiency (as described in Suemori et al. (2001) Dev. Dyn.

222:273-279). UndifFerentiated ES cells were maintained on a feeder layer (STO) of mitomycin C (WAKO, Osaka, Japan)-treated mouse embryonic fibroblasts in DMEM (SIGMA, St. Louis, MO)/F-12 (Invitrogen Coφ., Carlsbad, CA) supplemented with

0.1 mM 2-mercaptoethanol (SIGMA)/ 1,000 units/ml leukemia inhibitory factor (LEF;

Chemicon, Temecula, CA)/ 20% knockout serum replacement (Invitrogen Cθφ.)/4 ng/ml fibroblast growth factor-2 (FGF-2) (Upstate Biotechnology, Lake Placid, NY).

Subculturing of ES cells was performed by using 0.25%> trypsin (Invitrogen Coφ.) in PBS with 20% knockout serum replacement (Invitrogen Coφ.) and 1 μM CaCl₂

(WAKO) (as described in Kawasaki et al. (2002) Proc. Natl. Acad. Sci. USA 99:1580-

1585; Suemori et al. (2001) ev. Dyn. 222:273-279).

Example 2: Induction of Neural Progenitors from Primate ES Cells Primate ES cells were induced into neural progenitors by the SDIA method

(Kawasaki et al. (2002) Proc. Natl. Acad. Sci. USA 99:1580-1585). PA6 cells were plated on type I collagen-coated chamber slides (Becton Dickinson Labware, Franklin Lakes, NJ) or gelatin (SIGMA) -coated dishes (Becton Dickinson Labware) and used as a feeder cell layer. To strictly avoid contamination by incidentally differentiating cells, differentiated ES cell colonies with stem cell-like moφhology (tightly packed cells with a high nucleus/ cytoplasm ratio) were manually selected. Undifferentiated ES cell colonies were first washed twice with GMEM medium (SIGMA) supplemented with 10%> knockout serum replacement/ 1 μM pyruvate (SIGMA) / 0.1 μM nonessential amino acids (Invitrogen Coφ.)/ 0.1 μM 2-mercaptoethanol (SIGMA). After trypsinization for 5 min at 37 °C, partially dissociated ES cell clumps (10-50 cells/ clump) were plated on PA6 cells at a density of 1000 clumps/ 10-cm dish and cultured in the differentiation medium for 2 weeks. Monkey ES cells cultured on PA6 cells as outlined above were analyzed over time for expression of markers of neural progenitors. The clusters of cells began to be immunoreactive for NCAM and Musashi-1 within 3 days. NCAM and Musashi-1- positive cells increased in number until 2 weeks after stromal cell-derived inducing activity (SDIA) treatment (Kawasaki et al. (2000) Neuron 28:31-40) (Figure 1). At 2 weeks, 78.3±7.5% and 75.0±15.4% of the cells in the colonies were immunoreactive on PA6 cells with antibodies against NCAM and Musashi-1, respectively. The percentages of immunoreactive colonies were the highest at 2 weeks after SDIA treatment (NCAM=97.5±4.6%, Musashi-1=90.4±7.5%; Figure 1). Next, neurosphere-like neuroprogenitors from monkey ES cells were generated. The differentiated ES cell colonies (2 weeks after SDIA treatment) were detached from the feeder-layer cells by using a papain dissociation system (Worthington Biochemical Coφoration, Lakewood, NJ). Isolated colonies were cultured as floating spheres in neurobasal medium (Invitrogen Coφ.) with B27 supplement (Invitrogen Coφ.), 20 ng/ml FGF-2, 20 ng/ml epidermal growth factor (EGF; R&D Systems, Minneapolis, MN), and 10 ng/ml LIF for 1 week.

Interference differential microscopic imaging (IDMI) of the spheres generated from the ES cells revealed that they had a human neurosphere-like moφhology (data not shown).

Example 3: Differentiation of Neural Progenitors After 1 week of culturing the spheres induced from monkey ES cells on neurobasal medium comprising FGF-2, EGF, and LIF, the floating spheres were manually picked up and plated on ornithin-laminin-coated slides in neurobasal medium containing 20 ng/ml brain-derived neurotrophic factor (BDNF; SIGMA), 20 ng/ml neurotrophin-3 (NT3; SIGMA) and 10 ng/ml LIF. The spheres began to differentiate in response to BDNF and NT3, and were immunoreactive for Musashi-1 and NCAM antibodies (data not shown). One week after the plating, the differentiated spheres were stained with antibodies against TuJl, GFAP, Galactocerebroside C (GalC), Map2ab, GABA, glutamate (Glu), serotonin (Ser), or choline acetyltransferase (ChAT) (data not shown). The stained spheres showed mature neuron-like moφhology and expressed the postmitotic neuronal marker

TuJl (52.8±16.0%/D API), Map2ab(38.3±7 5 %/DAPI), GFAP (28.6±17.6 %/DAPI), and GalC(0.6±0.4 %/DAPI) (Figure 2A). Further, neurotransmitter expression was analyzed. The differentiated cells were immunopositive for GABA (28.6±10.7 %/TuJl), ChAT (43.0±20.0%/TuJl), TH (7.1±5.3%/TuJl), serotonin (3.3±1.7%/TuJl), and glutamate (4.3±5.3 %>/TuJI) (Figure 2B). These results indicate that the spheres were multipotential and consisted of neural stem cells.

After 1 week, the resulting spheres were fixed with 4% aldehyde (SIGMA), maintained in culture, or used for further experiments. It is notable that even 4 months after being cultured as spheres, the spheres from ES cells could differentiate into TuJl -positive, GFAP-positive, and GalC-positive cells (data not shown).

Example 4: Expansion of Dopaminergic Neurons from ES-derived Neural Progenitors The effect of FGF-20 on expansion of TH-positive cells for transplantation therapy of Parkinson's disease was analyzed (Figure 3). ES cell-derived spheres obtained as outlined in Examples 1-2 above were cultured as in Example 2 with various concentrations (1 pM, 10 pM, and 1 nM) of FGF-20, and also cultured in the presence of FGF-2, FGF-2 + EGF, FGF-2 + FGF-20, FGF-20, or FGF-2 + FGF-20 + EGF. In this manner, the spheres induced from monkey ES cells were plated on ornithin-laminin-coated slides as noted above and cultured with the various combinations of FGF-2, FGF-20, and/or EGF. One week later, the differentiated spheres were stained with anti-tyrosine hydroxylase (TH). TH-immunopositive cells were detected among the differentiated monkey ES cells (data not shown).

FGF-20 caused a significant increase in the number of TH-positive neuron cells in the presence of FGF-2 when compared to the number of TH-positive cells in the presence of either FGF-2 or FGF-20 alone (Figure 3). In the presence of EGF and FGF-2, the number of TH-positive cells was less than that observed for FGF-2 alone, or FGF-2 + FGF-20. In the presence of FGF-2 and EGF, FGF-2 could not increase the number of TH-positive cells. Thus the greatest number of TH-positive neuron cells was observed in the presence of FGF20 and FGF-2. FGF-20 also increased the rate of TH differentiation in a concentration-dependent manner (1 pM=3.8±1.8%/TuJl, 10 pM=8.8±4.8°/TuJl, 1 nM=24.3±9.8%/TuJl) in the presence ofFGF-2. Example 5: Transplantation of Dopaminergic Neurons from ES Cell-Derived Neural Progenitors In order to assess the functionality of dopaminergic neurons obtained from embryonic stem (ES) cell-derived neural progenitors, an MPTP primate model of Parkinson's disease was used. MPTP is a neurotoxin and can induce Parkinson-like symptoms in rodents and primates. The following protocols were used in this study.

Preparation of Animal Model of Parkinson's Disease

Adult male cynomolgus monkeys (Macaca fascicularis) weighing 2.5 - 3.5 kg were given intravenous injection of MPTP HCl (0.4mg/kg as free base) twice a week until persistant Parkinsonian behavior disturbances such as tremor, bradykinesia, and impaired balance were shown. The animals that presented stable Parkinsonism for over twelve weeks were used for transplantation of dopaminergic neurons obtained from ES cell-derived neural progenitors. All animals were fed with commercial pellets and fresh fruits and had free access to clean water. The monkeys were cared for and handled according to the Guideline for Animal Experiments of Kyoto University and the Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources (ILAR), Washington, D.C., USA.

Transplantation

The monkeys were anesthesized with pentobarbital (7.5 mg/kg, i.m., Dainippon Pharmaceutical; Osaka, Japan) and ketamine (10 mg/kg, Sankyo Co., Tokyo, Japan), and fixed in a surgical frame (Narishige, Tokyo, Japan). Monkey ES cells cultured in two 6-cm dishes (150,000-300,000 cells) were collected for each transplantation. The cells for transplantation represented a population of differentiated ES cells that contained a high percentage of TH-positive cells, prepared as described in Examples 1-4 above. Three days prior to the transplantation, BrdU (5 μg/ml; SIGMA) was added to the medium.

Using a stereotactic frame (Narishige) and injector (Muromachi Kikai Co., Tokyo, Japan), donor cells were transplanted into 3 target points in the putamen according to MRI findings and a Macaca fascicularis brain atlas (Szabo et al. (1984)

J. Comp Neurol. 222:265-300; Martin & Bowden (2000) Primate Brain Maps:

Structure of the Macaque Brain (Elsevier Science). After the surgery, the animals were given antibiotics for 1 week and a daily immunosuppressant (cyclosporin A, 10 mg/kg; i.m.; Carbiochem, San Diego, CA) until time of sacrifice. Weekly blood analyses were performed to check the dose of cyclosporin A.

Behavioral Assessment

Parkinsonian behavior was evaluated using a rating scale previously proposed by Akai et al. (1995) J. Pharmacol. Exp. Ther. 273:309-314, with a slight modification (see Table 1 below). Assessments with this scale were carried out by one examiner, who was unaware of the transplantation procedure used for each animal.

Magnetic Resonance Imaging (MRf)

Animals were submitted to a magnetic resonance imaging (MRI) examination on a 3.0 Tesla SIGMA system (General Electric, Milwaukee, WI). Animals were anesthesized by an intramuscular injection with ketamine hydrochloride (15 mg/kg) and xylazine (1.5mg/kg, Boehringer Ingelheim Vetmedica, St. Joseph, MI) and positioned into the magnet by using an MR-compatible headholder. TI -weighted images were used for further examinations.

Positron Emission Tomography (PET) A portion of the animals in cell-transplanted or sham-operated groups

1 R underwent PET scans using F-fluorodopa for in vivo evaluation of dopaminergic function under generalized anesthesia with continuous infusion of propofol (4mg/kg/hr, Zeneca Pharmaceuticals, Wilmington, DE) and vecuronium-bromide (0.25 mg/kg/hr Boehringer Ingelheim Vetmedica). Scans were performed on the ECAT EXACT HR PET scanner (Siemens-CTI, Knoxville, TE) at Bio-Functional Research Institute at National Cardio- Vascular Center (NCVC). Ethical permission for PET studies was obtained from the animal ethical committee of the NCVC. After intravenous injection of 185MBq of ¹⁸F-fluorodopa, data acquisition of brain radioactivity was taken for the subsequent 90 min. with animals receiving carbidopa (1 Omg/kg) 30 minutes prior to PET scan.

The following physiological parameters were monitored during the experiments: end-tidal carbon dioxide level, arterial gas analysis, blood pressure, heart rate and body temperature. Animals were heated with controlled heating blankets. Parametric images of dopamine irreversible metabolic rate of Ki (min^"1; considered a parameter for presynaptic dopaminergic function) was generated using time-radioactivity course in each voxel by multiple-time- graphical-analysis (Patlak et al. (1985) J. Cereb. Blood Flow Metab. 5:584-590), as a reference region taken in the bilateral occipital lobes. The F-fluorodopa Ki image was co-registered to the corresponding TI -weighted magnetic resonance image, obtained by IR-FSPGR sequence (TR=9.4, TI= 600, TE= 2.1 in msec) using 3-Tesla MRI scanner (Signa LX VH/i, GE, MI) and realigned to a standard space of Macaca fascicularis (Martin and Bowden (2000) Primate Brain Maps: Structure of the Macaque Brain (Elsevier Science). Dopaminergic function was evaluated by visual inspection of Ki images and by quantitative Ki values in the bilateral dorsal striatum determined by the corresponding MRI image.

Immunocytochemistrv and Immunohistochemistry For immunocytochemistry, cells were fixed with 4% paraformaldehyde for 10 min. After deep anesthesia was achieved with pentobarbital, the animals were transcardially perfused with 4% paraformaldehyde. The removed brains were fixed with 4% paraformaldehyde for 6 hr and cut frozen with a microtome at a 50-μm thickness. Immunohistochemical studies were performed using the free-floating method.

The slices or slides were first incubated in 0.3% Triton X and 5%o skim milk in PBS for 30 min. Then, they were incubated with antibodies against NCAM (Chemicon), Musashi-1 (a gift from Dr. H. Okano, Keio University, Japan), TuJl, (BabCO, Richmond, CA), GFAP (Chemicon), Map2ab (SIGMA), Galactocerebroside C (GalC, Chemicon), glutamate (Chemicon), chorine acetyltranspherase (ChAT, Chemicon), GABA (SIGMA), BrdU (Becton Dickinson), tyrosine hydroxylase (TH, Chemicon), serotonin (Dia Sorin, Stillwater, MN), dopamine transporter (DAT, Chemicon), or dopamine beta hydroxylase (DBH, Santa Cruz Biotechnology, Santa Cruz, CA) in 2% skim milk in PBS overnight at 4°C. After 3 rinses with PBS, they were incubated with FITC-labeled anti-mouse (Jackson Immunoresearch, West Grove, PA), Cy3- labeled anti-rabbit antibodies (Jackson Immunoresearch) as secondary antibodies for

1 hour at room temperature. After having been washed with PBS, the slices were mounted and analyzed. Results

The behavior of the postoperative five monkeys was analyzed based on their neurological score (see Table 1 below). As a result, 3 of the 6 animals that had received the ES cell-derived neural progenitors showed improvement. One animal became worse, and the other 2 showed no change in their behavioral score. Among the sham-operate animals, one animal showed improvement, and two animals showed no change. The postoperative mean behavior scores were significantly lower in ES cell-transplanted than in sham-operated monkeys (Figure 4A). Positron emission tomography (PET) revealed that ¹⁸F-fluorodopa uptake was upregulated in the behaviorally improved animal (Figure 4B). Sham-operated animals showed no improvement in this uptake.

Table 1. Neurological scores of MPTP-treated monkeys.

Behavior Scores

Alertness normal, 0; reduced, 1; absent, 2

Head checking movement present, 0; reduced, 1 ; absent, 2

Eyes normal, 0; reduced blinking, 1 ; eye closed, 2

Posture normal, 0; mildly abnormal, 1 ; abnormal, 2; grossly abnormal, 3

Balance normal, 0; impaired, 1 ; frequent falling, 2; no movement, 3

Motility, at rest normal, 0; mild bradykinesia, 1 ; bradykinesia, 2; akinesia, 3

Motility, reaction to ext. normal, 0; mildly reduced, 1; reduced, 2; absent, 3 stim.

Walking normal, 0; mildly reduced walking, 1; reduced walking, 2; no walking,

3

Tremor absent, 0; mild/not always, 1; moderate, 2; severe, 3

At 14 weeks after transplantation, the behaviorally improved animals were sacrificed and analyzed immunohistochemically. In ES cell-transplanted monkeys, BrdU-positive cells (85,000±25,166/mm³) were detected (using interference differential microscopic images (IDMI)) in the entire lateral striatum (data not shown). Of these cells, 66.7±12.1% were TuJ-1 positive. Tyrosine hydroxylase (TH)-immunopositive cells were also distributed throughout the entire striatum (data not shown). TH-immunoreactive cells (45,305±13,413/m³) were detected mainly in the marginal lesion of the graft, and 53.3±8.2 %> of the BrdU-positive cells were also immunopositive for TH (data not shown). BrdU-positive cells were also co-labeled with antibody to DAT. DAT-positive cells were distributed through the entire graft (data not shown). 50.0±12.6%> of the BrdU-positive cells were also immunoreactive with anti-DAT (data not shown). MRI findings of monkeys with transplanted ES cells reflected these distribution patterns (data not shown).

In contrast, no BrdU-positive cells were recognized in the lateral striatum of the sham-operated animals. In addition, Ki-67 staining indicated no Ki-67-positive cells in the graft (data not shown).

Conclusion

The results of these studies show that a highly enriched population of proliferating neural progenitors can be derived from monkey ES cells using the SDIA method. The resulting neurospheres can be easily cultured and expanded as a source of multipotent neural progenitors. Furthermore, a large number of dopaminergic neurons can be induced from ES cell-derived neural progenitors. FGF-20 in the presence of FGF-2 can expand the number of these dopaminergic neurons.

Transplantation of these cells into the striatum also attentuated MPTP-induced Parkinsonian symptoms. This result implies that not only dopaminergic but also multifunctional neurons may be generated from the transplanted cells. Thus, these studies confirm that these cells could differentiate into cholinergic, dopaminergic, glutaminergic, and GABAnergic neurons. In the event of ischemia, trauma, or hemorrhage, a large number of various neuron types are lost, and thus neural progenitors from ES cells have the potential to replace various neurons lost after these insults.

Functional imaging is available as a tool for in vivo assessment of dopaminergic neuronal differentiation, graft survival, and functional integration. PET imaging of presynaptic markers such as ¹⁸F-fluorodopa, fluorometatyrosine, or CFT is used to determine whether implanted cells in vivo have the molecular machinery necessary for dopamine synthesis and/or storage (Elsworth et al. (1994) Exp. Neurol.

126:00-304; Poyot et al. (2001) J. Cereb. Blood Flow Metab. 2001 :21:782-792; and

Doudet et al. (1998) Synapse. 29:225-232). In the present study, ¹⁸F-fluorodopa uptake after transplantation was assessed using PET. The results indicate that the transplanted cells functioned as dopaminergic neurons. There was not a significant difference in mean Ki value from entire putamen between the transplanted monkeys and sham-control monkey. However behaviorally improved animals showed increased Ki value in transplanted putamen. It is noteworthy that these animals were sacrificed at 3 months after transplantation, which may not have been sufficient time to recognize significant improvement in the mean Ki value from entire putamen. Other transplantation studies report that an improved Ki value from entire putamen was observed by 6 months after transplantation (Freed et al. (2001) New Engl. J. Med. 344:710-719) and that striatal uptake of fluorodopa was unchanged 5 to 6 months postoperatively but was markedly increased at 12 to 13 months and at 22 to24 months in patients who received fetal mesencephalic grafts (Widner et al. (1992) New Engl. J. Med. :321: 1556-1563).

To expand dopaminergic neurons from monkey ES cell-derived neuroprogenitors, various neurotrophic factors were tested. Ascorbic acid, sonic headgehoff (SHH), and EGF could not increase the number of TH-immunopositive cells (data not shown), whereas FGF20 did. F20 is a newly recognized member of the FGF family that is highly expressed in the brain, particularly in the substantia nigra (Ohmachi et al. (2000) Nat. Biotechnol. 18:675-679). The expression profile of FGF- 20 is quite different from that of other FGFs, indicating that FGF-20 plays a unique role in the brain. A previous study showed that recombinant FGF-20 enhances the survival of dopaminergic eurons (Ohmachi et al. (2000) Nat. Biotechnol. 18:675-679).

The results presented herein above indicate that FGF-20 may promote the differentiation of dopaminergic neurons. However, its effect is significant only in the presence of FGF-2, thus suggesting that both factors may be necessary for dopaminergic neuron differentiation. FGF-2 and EGF play a different role in the differentiation of neural precursors (Ciccolini and Svendsen (1998) J. Neurosci. 18:7869-7880). An earlier report showed that FGF-2 promoted neuronal differentiation, and EGF promoted glial differentiation, in vivo (Kuhn et al. (1997) J. Neurosci. 17:5820-5829). As shown herein above, for ES cells, EGF decreased dopaminergic neuronal differentiation, and FGF-20 had its maximum effect in the presence of FGF-2. These data further indicate that FGF-20 may induce dopaminergic differentiation from neuronal progenitors and/or promote the survival of these dopaminergic neurons.

As for the behavioral analysis, significant behavioral improvement was detected in the transplant-treated monkeys. According to the results of the PET study, an improvement in fluorodopa uptake was found in the behaviorally improved animals. The immunohistochemical analysis showed abundant TH-positive cell survival in the target area. In addition, the improvement in behavior was reasonable with that reported previously (Freed et al. (2001) New Engl. J. Med. 344:710-719; Lindvall et al. (1994) Ann. Neurol. 35:172-180; Bjorklund et al. Proc. Natl. Acad. Sci. USA 99:2344-2349).

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incoφorated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incoφorated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

THAT WHICH IS CLAIMED:

1. A method of treating a central nervous system (CNS) disorder in a mammal, said method comprising co-administration of a therapeutically effective amount of FGF-2 or variant thereof and a therapeutically effective amount of FGF-20 or variant thereof to said mammal, wherein said co-administration provides for an improvement in one or more symptoms associated with said CNS disorder.

2. The method of claim 1, wherein said mammal is a human.

3. The method of claim 2, wherein said FGF-2 is human FGF-2 or biologically active variant thereof having at least 70%> sequence identity to human FGF-2, and wherein said FGF-20 is human FGF-20 or biologically active variant thereof having at least 70% sequence identity to human FGF-20.

4. The method of claim 3, wherein at least one of said FGF-2, said FGF- 20, and said biologically active variants thereof is recombinantly produced.

5. The method of claim 1, wherein the improvement in said symptom is greater than an improvement in said symptom that would be observed with administration of said FGF-2 or variant thereof alone or with administration of said FGF-20 or variant thereof alone.

6. The method of claim 1, wherein said FGF-2 or variant thereof and said FGF-20 or variant thereof are administered simultaneously as a single pharmaceutical composition.

7. The method of claim 1, wherein said FGF-2 or variant thereof and said FGF-20 or variant thereof are administered simultaneously or sequentially as two separate pharmaceutical compositions.

8. The method of claim 1, wherein said co-administration comprises a single administration of said FGF-2 or variant thereof and said FGF-20 or variant thereof.

9. The method of claim 1, wherein said co-administration comprises multiple administrations of said FGF-2 or variant thereof and said FGF-20 or variant thereof.

10. The method of claim 1, wherein said CNS disorder is a neurodegenerative disorder.

11. The method of claim 10, wherein said neurodegenerative disorder is selected from the group consisting of motor neuron disease, multiple sclerosis, muscular dystrophy, diabetic neuropathy, demyelinating peripheral neuropathies, Parkinson's disease, Alzheimer's disease, Huntington's disease, Korsakoff s disease, Down's syndrome, and a sequela of chronic epilepsy.

12. The method of claim 10, wherein said neurodegenerative disorder is selected from the group consisting of a sequela of traumatic central nervous system injury, a sequela of stroke, and a sequela of ischemia.

13. The method of claim 1, wherein said FGF-2 or variant thereof and said FGF-20 or variant thereof are administered by a route of administration selected from the group consisting of parenteral, intrathecal, intracerebral, transmucosal, topical, transdermal, inhalation, and intranasal.

14. The method of claim 13, wherein said route of administration provides for administering directly to a tissue innervated by the trigeminal nerve, the olfactory nerve, or both the trigeminal and olfactory nerves.

15. The method of claim 1, wherein said mammal also receives cell transplantation therapy within a region of the CNS.

16. The method of claim 15, wherein said cell transplantation therapy comprises transplantation of a population of neural progenitor cells within said region of the CNS.

17. The method of claim 15, wherein said cell transplantation therapy comprises transplantation of a population of dopaminergic neurons within said region of the CNS.

18. The method of claim 17, wherein said dopaminergic neurons are derived from embryonic stem cells.

19. A method of treating a central nervous system (CNS) disorder in a mammal in need of treatment thereof, said method comprising: a) obtaining a population of neural progenitor cells; b) culturing said population of neural progenitor cells in a culture medium that provides for proliferation of said neural progenitor cells, wherein said culture medium comprises FGF-2 or variant thereof and FGF-20 or variant thereof, whereby a population of differentiating neural progenitor cells is obtained; and c) transplanting said population of differentiating neural progenitor cells into a target site within the CNS of said mammal.

20. The method of claim 19, wherein said FGF-2 or variant thereof and said FGF-20 or variant thereof are present in said culture medium in amounts effective to promote differentiation of dopaminergic neurons from said neural progenitor cells, whereby the population of differentiating neural progenitor cells is enriched in dopaminergic neurons.

21. The method of claim 19, wherein said CNS disorder is a neurodegenerative disorder.

22. The method of claim 21, wherein said neurodegenerative disorder is selected from the group consisting of motor neuron disease, multiple sclerosis, muscular dystrophy, diabetic neuropathy, demyelinating peripheral neuropathies, Parkinson's disease, Alzheimer's disease, Huntington's disease, KorsakofPs disease, Down's syndrome, a sequela of traumatic central nervous system injury, and a sequela of chronic epilepsy.

23. The method of claim 19, wherein said target site resides within a region of the basal ganglia.

24. The method of claim 23, wherein the population of differentiating neural progenitor cells is enriched in dopaminergic neurons.

25. The method of claim 24, wherein said CNS disorder is Parkinson's disease.

26. The method of claim 19, wherein said neural progenitor cells are of human origin.

27. The method of claim 26, wherein said neural progenitor cells are derived from human embryonic stem cells.

28. The method of claim 19, wherein said mammal is a human.

29. The method of claim 28, wherein said FGF-2 is human FGF-2 or biologically active variant thereof having at least 70% sequence identity to human FGF-2, and wherein said FGF-20 is human FGF-20 or biologically active variant thereof having at least 70% sequence identity to human FGF-20.

30. The method of claim 29, wherein at least one of said FGF-2, said FGF- 20, and said biologically active variants thereof is recombinantly produced.

31. The method of claim 19, further comprising co-administration of a therapeutically effective amount of FGF-2 or variant thereof and a therapeutically effective amount of FGF-20 or variant thereof to said mammal wherein said coadministration occurs prior to, simultaneously with, or following transplantation of said population of differentiating neural progenitor cells.

32. The method of claim 31, wherein said mammal is a human, and wherein said FGF-2 is human FGF-2 or biologically active variant thereof having at least 70%) sequence identity to human FGF-2, and wherein said FGF-20 is human FGF-20 or biologically active variant thereof having at least 70%> sequence identity to human FGF-20.

33. The method of claim 32, wherein at least one of said FGF-2, said FGF-

20, and said biologically active variants thereof is recombinantly produced.

34. The method of claim 31 , wherein said FGF-2 or variant thereof and said FGF-20 or variant thereof are administered simultaneously as a single pharmaceutical composition.

35. The method of claim 31 , wherein said FGF-2 or variant thereof and said FGF-20 or variant thereof are administered simultaneously or sequentially as two separate pharmaceutical compositions.

36. The method of claim 31 , wherein said FGF-2 or variant thereof and said FGF-20 or variant thereof are administered by a route of administration selected from the group consisting of parenteral, intrathecal, intracerebral, transmucosal, topical, transdermal, inhalation, and intranasal.

37. The method of claim 36, wherein said route of administration provides for administering directly to a tissue innervated by the trigeminal nerve, the olfactory nerve, or both the trigeminal and olfactory nerves.

38. The method of claim 31, wherein said co-administration comprises a single administration of said FGF-2 or variant thereof and said FGF-20 or variant thereof.

39. The method of claim 31 , wherein said co-administration comprises multiple administrations of said FGF-2 or variant thereof and said FGF-20 or variant thereof.

40. The method of claim 31, wherein said CNS disorder is a neurodegenerative disorder.

41. The method of claim 40, wherein said neurodegenerative disorder is selected from the group consisting of motor neuron disease, multiple sclerosis, muscular dystrophy, diabetic neuropathy, demyelinating peripheral neuropathies, Parkinson's disease, Alzheimer's disease, Huntington's disease, Korsakoff s disease, Down's syndrome, and a sequela of chronic epilepsy.

42. The method of claim 40, wherein said neurodegenerative disorder is selected from the group consisting of a sequela of traumatic central nervous system injury, a sequela of stroke, and a sequela of ischemia.

43. A method for promoting growth, proliferation, differentiation, or survival of a cell within a target site of the central nervous system (CNS) of a mammal in need of treatment for a CNS disorder, said method comprising co- administering to said mammal a therapeutically effective amount of FGF-2 or variant thereof and a therapeutically effective amount of FGF-20 or variant thereof, wherein said FGF-2 and said FGF-20 are delivered to said target site in an amount effective to promote growth, proliferation, differentiation, or survival of said cell.

44. The method of claim 43, wherein said cell within said target site is an existing CNS cell selected from the group consisting of a neural progenitor cell and a dopaminergic neuron.

45. The method of claim 43, wherein said cell within said target site is a transplanted donor cell.

46. The method of claim 45, wherein said transplanted donor cell is selected from the group consisting of a neural progenitor cell and a dopaminergic neuron.

47. The method of claim 46, wherein said transplanted donor cell is derived from an embryonic stem cell.

48. The method of claim 43, wherein said mammal is a human.

49. The method of claim 48, wherein said FGF-2 is human FGF-2 or biologically active variant thereof having at least 70% sequence identity to human FGF-2, and wherein said FGF-20 is human FGF-20 or biologically active variant thereof having at least 10% sequence identity to human FGF-20.

50. The method of claim 49, wherein at least one of said FGF-2, said FGF-

20, and said biologically active variants thereof is recombinantly produced.

51. The method of claim 43, wherein said FGF-2 or variant thereof and said FGF-20 or variant thereof are administered simultaneously as part of a single pharmaceutical composition.

52. The method of claim 43, wherein said FGF-2 or variant thereof and said FGF-20 or variant thereof are administered simultaneously or sequentially as two separate pharmaceutical compositions.

53. The method of claim 43, wherein said FGF-2 or variant thereof and said FGF-20 or variant thereof are administered by a route of administration selected from the group consisting of parenteral, intrathecal, intracerebral, transmucosal, topical, transdermal, inhalation, and intranasal.

54. The method of claim 53, wherein said route of administration provides for administering directly to a tissue innervated by the trigeminal nerve, the olfactory nerve, or both the trigeminal and olfactory nerves.

55. The method of claim 43, wherein said co-administration comprises a single administration of said FGF-2 or variant thereof and said FGF-20 or variant thereof.

56. The method of claim 43, wherein said co-administration comprises multiple administrations of said FGF-2 or variant thereof and said FGF-20 or variant thereof.

57. The method of claim 43, wherein said CNS disorder is a neurodegenerative disorder.

58. The method of claim 57, wherein said neurodegenerative disorder is selected from the group consisting of motor neuron disease, multiple sclerosis, muscular dystrophy, diabetic neuropathy, demyelinating peripheral neuropathies, Parkinson's disease, Alzheimer's disease, Huntington's disease, Korsakoff s disease, Down's syndrome, and a sequela of chronic epilepsy.

59. The method of claim 57, wherein said neurodegenerative disorder is selected from the group consisting of a sequela of traumatic central nervous system injury, a sequela of stroke, and a sequela of ischemia.

60. A method for promoting differentiation of dopaminergic neurons from a population of neural progenitor cells, said method comprising: a) obtaining a population of neural progenitor cells that comprises at least one neural progenitor cell that is capable of differentiating into neurons and glia; and b) culturing said neural progenitor cells in a culture medium that provides for differentiation of said neural progenitor cells into neurons and glia, said culture medium comprising FGF-2 or variant thereof and FGF-20 or variant thereof, wherein said FGF-2 or variant thereof and said FGF-20 or variant thereof are present in said culture medium in amounts effective to promote differentiation of said dopaminergic neurons from said neural progenitor cells.

61. The method of claim 60, wherein said neural progenitor cells are of human origin.

62. The method of claim 61, wherein said neural progenitor cells are derived from human embryonic stem cells.

63. The method of claim 60, wherein said FGF-2 is human FGF-2 or biologically active variant thereof having at least 70% sequence identity to human FGF-2, and wherein said FGF-20 is human FGF-20 or biologically active variant thereof having at least 70% sequence identity to human FGF-20.

64. The method of claim 63, wherein at least one of said FGF-2, said FGF- 20, and said biologically active variants thereof is recombinantly produced.

65. The method of claim 60, wherein FGF-2 or variant thereof is present at a concentration of about 1 picamole (pM) to about 50 nanomole (nM), and wherein said FGF-20 or variant thereof is present at a concentration of about 1 pM to about 50 nM.

66. The method of claim 65, wherein said FGF-2 or variant thereof is present at a concentration of about 50 pM to about 1 nM, and wherein said FGF-20 or variant thereof is present at a concentration of about 100 pM to about 10 nM.

67. A composition comprising a population of differentiating neural progenitor cells enriched in dopaminergic neurons, said population being produced according to the method of any one of claims 60-66.

68. A pharmaceutical composition comprising a combination of FGF-2 or variant thereof and FGF-20 or variant thereof, wherein said combination renders said pharmaceutical composition effective for use in treating a central nervous system disorder.

69. The pharmaceutical composition of claim 68, wherein said FGF-2 is human FGF-2 or biologically active variant thereof having at least 70% sequence identity to human FGF-2, and wherein said FGF-20 is human FGF-20 or biologically active variant thereof having at least 10% sequence identity to human FGF-20.

70. The method of claim 69, wherein at least one of said FGF-2, said FGF- 20, and said biologically active variants thereof is recombinantly produced.

71. The pharmaceutical composition of claim 68, wherein said composition is suitable for co-administration of a therapeutically effective amount of said FGF-2 or biologically active variant thereof and a therapeutically effective of said FGF-20 or biologically active variant thereof, wherein said co-administration provides for an improvement in one or more symptoms associated with said CNS disorder.