WO2008115880A2 - Use of leptin for the treatment or prevention of parkinson's disease - Google Patents

Abstract

Disclosed are methods of treating or preventing Parkinson's disease or secondary parkinsonism in a subject, comprising administering to the subject a pharmaceutically effective amount of a leptin polypeptide, leptin transporter polypeptide, leptin receptor polypeptide, or a variant thereof, wherein administering the leptin polypeptide, leptin transporter polypeptide, leptin receptor polypeptide, or variant thereof results in treatment or prevention of Parkinson's disease or secondary parkinsonism in the subject.

Description

DESCRIPTION

USE OF LEPTIN FOR THE TREATMENT OR PREVENTION OF PARKINSON'S

DISEASE

This application claims the benefit of priority to U.S. provisional patent application Serial No. 60/896,202, filed March 21, 2007, the entire content of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to the fields of pharmacology and neurodegenerative diseases. More particularly, it concerns use of leptin polypeptides or variant leptin polypeptides in the treatment or prevention of Parkinson's disease. 2. Description of Related Art Parkinson's disease is one of the most common neurodegenerative disorders, affecting about 1% of the population over the age of 50 (Dawson and Dawson, 2003). It is characterized by progressive and selective loss of dopaminergic neurons in the substantia nigra (SN) pars compacta (SNc), which results in dopamine depletion causing rigidity, tremor, bradykinesia and postural imbalance. Current treatment of Parkinson's disease still largely relies on levodopa

(Katzenschlager and Lees, 2002; Olanow et al, 2006), which is effective in improving the motor dysfunction. However, the effectiveness of these drugs changes following disease progression.

Dopamine agonists represent another group of drugs for the treatment of motor symptoms in patients with Parkinson's disease (Jankovic, 2006). Since a major feature of Parkinson's disease is the progressive loss of dopaminergic neurons in the substantia nigra, besides treating the symptoms of the disease, a treatment that prolongs the survival of dopaminergic neurons would be beneficial in slowing down the progression of the disease.

With increased understanding of the pathogenesis of Parkinson's disease (Cardoso et al, 2005), varieties of potential neuroprotective strategies including anti-oxidants (Zhang et al, 2002; Beal, 2004), anti-apoptotic drugs, anti-inflammatory drugs (Gao et al, 2003) and other forms of therapy are being studied or evaluated. Neurotrophic factors, such as glia- derived neurotrophic factor (GDNF), also belongs to the neuroprotective category. GDNF has been shown to be effective in protecting the dopamine neurons in different animal models of Parkinson's disease (Kirik et al., 2004). BDNF is another neurotrophic factor that could possibly benefit the survival of dopamine neurons (Fumagalli et al, 2006).

Leptin (also known as "ob protein"), the 16 kDa product of the ob gene, was discovered by Zhang et al. in 1994 (Zhang et al., 1994). It is predominantly synthesized and expressed in adipose tissues (Zhang et al., 1994, Ji et al., 1998). It functions as a potent physiological signal in the regulation of body weight, energy expenditure, feed intake, adiposity, fertility and immune functions (Houseknecht et al., 1998, Lord et al., 1998, Garcia et al., 2002). Leptin deficient ob/ob mice suffer from morbid obesity, diabetes mellitus, and hyperlipidemia, while leptin administration to these mice results in reversal of these disorders (Halaas et al., 1995). However, the administration of leptin to humans suffering from morbid obesity failed to suppress appetite or reduce weight (Friedman et al., 2002). This disappointing result may stem from increased leptin levels in obese individuals, and a presumed leptin resistance (Mark et al., 2002; Berg et al., 2002). Although leptins have been described as anti-obesity agents that can be applied in the treatment of obesity in Parkinson's disease patients (see U.S. Patent 7,109,339 and U.S. Patent Application Pub. No. 2004/0077650), they have not been described as therapeutic agents that can be applied in the treatment or prevention of neurodegenerative diseases. Therapeutic use for leptin or leptin receptors include diabetes (see, e.g., WO 98/55139), WO 98/55139, WO 98/12224, and WO 97/02004), hematopoiesis (see, e.g., WO 97/27286 and WO 98/18486), infertility (WO 97/15322 and WO 98/36763), and tumor suppression (see, e.g., WO 98/48831).

In view of the limitations of existing therapies for Parkinson's disease, there is the need for therapies that are more effective, and that are targeted to the dopaminergic degeneration associated with Parkinson's disease. Further, there is the need for effective preventive measures to reduce the risk of development of Parkinson's disease.

SUMMARY OF THE INVENTION

The present invention provides for the use of leptin and variants of leptin in the treatment and prevention of Parkinson's disease and secondary parkinsonism. The present inventors have found that leptin, which is a circulating hormone that is able to cross the blood brain barrier, functions as a neurotrophic factor, and is involved in protecting dopaminergic neurons from neurodegeneration. The present invention generally pertains to a method of treating or preventing Parkinson's disease or secondary parkinsonism in a subject, comprising administering to the subject a pharmaceutically effective amount of a leptin polypeptide or a variant thereof, wherein administering the leptin polypeptide or variant thereof results in treatment or prevention of Parkinson's disease in the subject. The present invention also generally pertains to a method of treating or preventing Parkinson's disease in a subject, comprising administering to the subject a pharmaceutically effective amount of a leptin transporter polypeptide, leptin receptor polypeptide, or a variant thereof. The present invention further pertains to a method of treating or preventing Parkinson's disease or secondary parkinsonism in a subject that involve administering to the subject a pharmaceutically effective amount of a synthetic leptin receptor ligand or an agent that promotes of function of a leptin transporter to facilitate transport of leptin to the brain, especially the substantia nigra. A synthetic leptin receptor ligand is defined as an agent that activates a leptin receptor. A synthetic leptin receptor ligand may be a small molecule, a peptide, a polypeptide, an antibody, an antibody fragment, a DNA, or an RNA.

The subject can be any subject, such as a primate or laboratory animal such as mouse or rat. In particular embodiments, the subject is a human. The subject, for example, may be a subject who has been previously diagnosed with Parkinson's disease who is in need of treatment. In other embodiments, the subject is an individual who has not been diagnosed with Parkinson's disease, but who may be at risk of developing Parkinson's disease. For example, the subject at risk of developing Parkinson's disease may be a subject who has one or more neurological symptoms of Parkinson's disease which may not be of sufficient severity or specificity to warrant a diagnosis of Parkinson's disease. Alternatively, for example, the subject may be an individual with a family history of Parkinson's disease, who may or may not have a neurological deficit, but who is considered to be at risk of developing Parkinson's disease. The subject, for example, may have a gene which is believed to increase the likelihood of developing Parkinson's disease (e.g., one of PARKl -P ARK9, as discussed further below).

In certain embodiments, the method is further defined as a method of reducing the rate of neuronal cell degeneration in a subject. More particularly, the neuronal cell degeneration may be dopaminergic neuronal degeneration. In some embodiments, the method further comprises assessing the severity of neuronal degeneration in a subject. This may be performed using any method known to those of ordinary skill in the art. For example, it may involve assessing the severity of any of the symptoms associated with Parkinson's disease before and after administration of the leptin polypeptide or variant. Thus, in some embodiments, administering the leptin polypeptide or variant thereof results in improvement of one or more motor deficits in the subject.

Administration of a leptin polypeptide, a leptin transporter polypeptide, a leptin receptor polypeptide, or a variant thereof can be by any method known to those of ordinary skill in the art. Many such methods of administration are known in the art, and all are contemplated for application in the present methods. For example, administering may be further defined as intravenous administration, intracerebral administration, administration into the cerebrospinal fluid, intraspinal administration, epidural administration, intralesional administration, topical administration, intraarterial administration, oral administration, regional administration, local administration, systemic administration, intraperiotoneal administration, intratumoral administration, intrathecal administration, or administration via an implantable delivery device. In a particular embodiment, administration is further defined as intravenous administration. In another particular embodiment, administration is further defined as intracerebral administration. Intracerebral administration can be by any method known to those of ordinary skill in the art. For example, intracerebral administration may be via direct injection, or administration using a drug delivery device. In some embodiments, the drug delivery device is an implantable drug delivery device. In a particular embodiment, the implantable drug delivery device is an osmotic minipump. In some embodiments, a leptin polypeptide, a leptin transporter polypeptide, a leptin receptor polypeptide, or a variant thereof is provided to the subject by administering to the subject a nucleic acid sequence encoding a leptin polypeptide, a leptin transporter polypeptide, a leptin receptor polypeptide, or a variant thereof. In particular embodiments, the nucleic acid is comprised within an expression construct, and wherein the nucleic acid sequence is under the transcriptional control of a promoter. Nucleic acid delivery techniques involving expression constructs are well-known to those of ordinary skill in the art, and are discussed in greater detail in the specification below. For example, the nucleic acid may include polynucleotide that encodes a polypeptide that comprises at least 10 contiguous amino acids of SEQ ID NO:1. In some embodiments, the expression construct is a viral vector. For example, the viral vector may be an adenovirus vector, an adeno-associated virus vector, a herpesvirus vector, a retrovirus vector, a lentivirus vector, a vaccinia virus vector, or a polyoma vector. In particular embodiments, the viral vector is an adenovirus vector. In further embodiments, the nucleic acid is comprised within a liposome. In some embodiments, the method further comprises identifying a subject in need of treatment or prevention of Parkinson's disease. Identification can be by any method known to those of ordinary skill in the art. For example, by clinical examination, by genetic screening, and so forth. In further embodiments, the method further comprising measuring a response to therapy following administration of the leptin polypeptide, leptin transporter polypeptide, leptin receptor polypeptide, or variant thereof. Measuring a response to therapy can be by any method known to those of ordinary skill in the art. For example, it may involve measuring or grading the severity of one of the symptoms of Parkinson's disease. In some examples, it involves assessment of motor function of the subject before and after administering the leptin polypeptide, leptin transporter polypeptide, leptin receptor polypeptide, or variant thereof.

In particular embodiments of the present invention, the methods involve administering one or more secondary forms of therapy to the subject for the treatment or prevention of Parkinson's disease. The secondary form of therapy can be any therapy or measure for the treatment or prevention of Parkinson's disease known to those of ordinary skill in the art. For example, the secondary form of therapy may be selected from the group consisting of pharmacological therapy, growth factors, gene therapy, immunotherapy, surgical therapy, cell transplantation therapy, herbal medicines, acupuncture, complementary therapies such as yoga and Tai Chi, therapeutic massage, and dietary supplements. In particular embodiments, the secondary form of therapy is pharmacological therapy. For example, the pharmacological therapy may be levodopa, a dopamine agonist, selegiline, an anticholinergic, amantadine, a catechol 0-methyltransferase (COMT) inhibitor, an anti-inflammatory agent, an antioxidant, an anti-apoptotic agent, a neurotrophic factor, herbal medicines, a coenzyme Q-IO, or a ubiquinone. The leptin polypeptide or variant thereof may be administered prior to, currently with, or following administration of the secondary form of therapy. In some embodiments, a secondary pharmacological agent for the treatment or prevention of Parkinson's disease is administered by formulating it in a single composition with the leptin polypeptide or variant leptin polypeptide. In further embodiments, the secondary agent and leptin polypeptide or variant leptin polypeptide are formulated in separate pharmaceutical compositions. The dose of leptin polypeptide or variant thereof can be any dose known or suspected by those of ordinary skill in the art to be of benefit in the treatment or prevention of Parkinson's disease. For example, the leptin polypeptide or variant thereof may be administered at a dose of about 0.01 μg/kg/24 hr to about 1 g/kg/24hr. It may be administered, for example, in a single dose, or in divided doses, or by continuous infusion, as discussed in greater detail elsewhere in this specification. In more particular embodiments, the leptin polypeptide or variant thereof is administered at a dose of about 0.1 μg/kg/24 hr to about 500 mg/kg/24 hr. In even more particular embodiments, it is administered at a dose of about 0.5 μg/kg/24 hr to about 50 mg/kg/24 hr. In further embodiments, it is administered at a dose of about 0.2 mg/kg/24 hr to about 5 mg/kg/24 hr.

In particular embodiments, the method of treating or preventing Parkinson's disease involves administering to the subject a leptin polypeptide. Leptin polypeptides are discussed in greater detail in the specification below. For example, the leptin polypeptide may comprise at least 50 contiguous amino acids of SEQ ID NO:1. In more particular embodiments, the leptin polypeptide comprises at least 100 contiguous amino acids of SEQ ID NO: 1. In even more particular embodiments, the leptin polypeptide comprises at least 500 contiguous amino acids of SEQ ID NO:1. The leptin receptor polypeptide, for example, may comprise at least 50, at least 100, or at least 500 contiguous amino acids of SEQ ID NO: 14 or SEQ ID NO: 15. The leptin transporter polypeptide, for example, may comprise at least 50, at least 100, or at least 500 contiguous amino acids of SEQ ID NO: 105.

In other embodiments, the method involves administering variant leptin polypeptide, a variant leptin transporter polypeptide, or a variant leptin receptor polypeptide. Variant leptin polypeptides, leptin transporter polypeptides, and leptin receptor polypeptides are discussed at length in the specification below. The method set forth herein may involve administering to the subject a pharmaceutically effective amount of a composition comprising a leptin polypeptide or variant thereof and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be any carrier known to those of ordinary skill in the art. For example, the carrier may be an aqueous carrier. The composition may comprise any number of excipients known to those of ordinary skill in the art. Examples of carriers and excipients contemplated by the present methods are discussed at length below. The composition may further comprise one or more secondary therapeutic agents that can be applied in the treatment or prevention of Parkinson's disease. Examples of such agents include any of those secondary pharmacological agents discussed above. The present invention also generally pertains to kits that include at least one sealed vial, wherein the vial includes a leptin polypeptide or variant thereof. In some embodiments, the kit includes two or more sealed vials. The kit may optionally include a catheter or drug delivery device. For example, in some embodiments, the kit includes an osmotic mini-pump. It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention.

Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention.

The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternative are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device and/or method being employed to determine the value.

As used herein the specification, "a" or "an" may mean one or more, unless clearly indicated otherwise. As used herein in the claim(s), when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

The following figures form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1. Loss of tyrosine hydroxylase (TH) neurons in the substantia nigra of ob/ob mice. 4-month old ob/ob or wild type mice were perfused with 4% paraformaldehyde. Every fourth brain section from the substantia nigra region was used for performing immunohistochemistry with a rabbit-anti-TH antibody (1 :1000, Pelfreeze). TH- immunostaining was shown at two different levels (Panels A-B).

FIG. 2. TH immunoreactive neurons in mouse primary mesencephalon cultures treated with our without leptin (1 μM) and MPP+ (10 μM). FIG. 3. Leptin protected midbrain primary TH-neurons from MPP+ (10 μM) chalenge. Primary cultured cells in triplicate wells were pretreated with or without leptin as labed on the bars, and then treated with (+) or without (-) MPP+. TH immunoreactive cells were counted. FIG. 4. Protection of primary dopamine neurons by leptin from rotenone. Primary cultured cells in triplicate wells were pretreated with or without leptin (1 μM), and then treated with rotenone. *, p<0.05 and #, p<0.01 compared to the control cells without leptin.

FIG. 5. Body weight and food intake in mice treated with subcutaneous infusion of leptin (n=5) or saline (n=5). Mice were implanted with osmotic mini-pumps filled with leptin in saline or saline alone. Measurement of body weight and food intake was performed daily for 7 days before MPTP treatment. *p<0.01 compared to saline treated group.

FIG. 6. Rotarod performance of mice pretreated with saline or leptin and then challenged with MPTP or saline. Rotarod performance was tested on the day before MPTP challenge (day 0) and at day 2 and day 7 after MPTP challenge. D , saline pretreatment and saline challenge (n=5); ', leptin pretreatment and MPTP challenge (n=4), , saline pretreatment and MPTP challenge (n=5).

FIG. 7. Leptin protected TH-fϊber in the striatum of mice challenged with MPTP. 7 days after MPTP challenge, brain sections from these mice were used for immunohistochemistry to detect TH-fϊbers in the striatum. Sal/Sal, saline pretreatment and saline challenge (n=5); Lep/MPTP, leptin pretreatment and MPTP challenge (n=4); Sal/MPTP, saline pretreatment and MPTP challenge (n=5). The right panel shows the quantification of the density of the TH- fibers in the striatum of mice from different treatment groups.

FIG. 8A, 8B. Intermittent i.p. administration of leptin prevents the loss of dopaminergic fibers in the striatum induced by MPTP. FIG. 8A - Immunostaining of tyrosine hydroxylase (TH) for dopaminergic fibers in the striatum. FIG. 8B - Quantitative data of TH-immunoreactive fiber density in the striatum. Sal-Sal, saline pretreatment and saline challenge; SaI-MPTP, saline pretreatment and MPTP challenge; Lep-0.3 mg/kg-MPTP, pretreatment with leptin at 0.3 mg/kg, twice per day; Lep-3 mg/MPTP, pretreatment with leptin at 3 mg/kg, twice per day. N = 5 per group. ### P<0.001 compared to Sal-Sal; *P<0.05 compared to SaI-MPTP. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention is based on the finding that leptin polypeptides and variants thereof can be applied in treatment or preventing Parkinson's disease. In particular, the inventors have found that administration of leptin protects against dopaminergic neuro degeneration in an animal model.

A. Leptin Polypeptides, Leptin Transporter Polypeptides, Leptin Receptor Polypeptides, and Variants Thereof

The present invention pertains to use of leptin polypeptides or variants of leptin polypeptides for the treatment or prevention of Parkinson's disease.

Throughout this application, the term "leptin polypeptide" is intended to refer to a polypeptide comprising an amino acid sequence from a human lepin or an orthologous sequence from a different species (e.g., murine leptin), wherein the polypeptide maintains some or all of the function of the full-length leptin protein. A list of examples of leptin polypeptides includes SEQ ID NO:1 (Homo sapiens), SEQ ID NO:2 (Mus musculus), SEQ ID NO:3 (Bos taurus), SEQ ID NO:4 (Rattus norvegicus), SEQ ID NO:5 (Sus scrofa), SEQ ID NO:6 (Fugu rubripes), SEQ ID NO:7 (African clawed frog), SEQ ID NO:8 (rhesus monkey), SEQ ID NO: 9 (rainbow trout), and SEQ ID NO: 10 (rabbit).

The full-length amino acid sequence of human leptin is provided herein, and is designated SEQ ID NO:1. "Leptin polypeptide" as used herein refers to an amino acid sequence that is either the full-length leptin protein sequence or less than the full-length sequence of the protein, so long as the amino acid sequence maintains some or all of the function of the full-length sequence. The term "leptin polypeptide" as set forth herein also refers to polypeptides that optionally include one or more additional amino acids attached to the N-terminus or the C-terminus of the polypeptide sequence derived from the protein.

A "leptin polypeptide" refers to a consecutive amino acid segment of a full-length leptin protein that is of any length, including the full length sequence. For example, the leptin polypeptide can include at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, or more consecutive amino acids of a full-length leptin protein. One of ordinary skill in the art would understand how to generate a leptin polypeptide in view of the substantial information in the art regarding leptins, including the amino acid sequences, known in the art. Included within the definition of "leptin polypeptides" are leptin precursors, such as those set forth in GenBank Accession number NP 000221 (leptin precursor, Homo sapiens; SEQ ID NO: 11), P41159 (leptin precursor; SEQ ID NO: 12), and AAH69323 (leptin precursor, Homo sapiens, SEQ ID NO: 13). Examples of leptin receptor polypeptides include those sequences set forth in

GenBank Accession No. AAB09673 (Homo sapiens; SEQ ID NO: 14), AAC23650 (Homo sapiens; SEQ ID NO: 15), NP 036728 (Rattus norvegicus; SEQ ID NO: 16), NP 989654 (Gallus gallus; SEQ ID NO: 17), NP 001012285 (Bos taurus; SEQ ID NO: 18), NP 001019758 (Sus scrofa; SEQ ID NO: 19), and AAZ78136 (Xenopus laevis; SEQ ID NO:20). Leptin receptor (OBR) has six alternatively spliced forms (OBRa-f or LRa-f). The

OBRa is the short form of OBR corresponding to the OBR amino acid sequence 1-894. This isoform of leptin receptor acts as a leptin transporter. The sequence of OBRa is as set forth in SEQ ID NO:105.

A "variant" as used herein refers to an amino acid sequence wherein the polypeptide has one or more amino acid changes that are distinct from the wild-type polypeptide, but wherein the polypeptide retains an acceptable level of equivalent biological function. For example, it is well understood by the skilled artisan that, inherent in the definition of a "variant leptin polypeptide," is the concept that there is a limit to the number of changes that may be made within a defined portion of a molecule and still result in a molecule with an acceptable level of equivalent biological activity, e.g., ability of function as a leptin. "Variant leptin polypeptide" is thus defined herein as any leptin polypeptide in which some, or most, of the amino acids may be substituted as compared to a polypeptide derived from a leptin protein so long as the polypeptide retains substantially similar leptin activity in the context of the uses set forth herein. An amino acid sequence of any length is contemplated within the definition of a variant, so long as the polypeptide retains an acceptable level of equivalent biological activity.

Any of the leptin polypeptides, leptin transporter polypeptides, leptin receptor polypeptides, or variants thereof may include at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, or more consecutive amino acids. Further, any of the polypeptides described herein may include one or more additional amino acids at either the C-terminal or N-terminal end that are not required for function of the polypeptide as a leptin, a leptin transporter, or a leptin receptor. For example, a leptin polypeptide may include a total of greater than 1000, 500-1000, 400-499, 300-399, 200-299, 100-199, 80-99, 60-79, 50-59, 40- 49, 30-39, 20-29, 10-19, 9, 8, 7, 6, 5, or 4 amino acid residues, as long as there remains an acceptable level of leptin biological activity.

Of course, a plurality of distinct proteins/polypeptides/peptides with different substitutions may easily be made and used in accordance with the invention. Additionally, in the context of the invention, a variant can be a homolog or paralog from any species or organism. One of ordinary skill in the art will understand that many variant polypeptides would likely exist and can be identified using commonly available techniques. Examples of variant leptin polypeptides include the following: (1) a silent mutation at codon 25 (CAA/CAG, glutamine); a silent mutation at codon 102(AAC/ AAT) in humans (Li et al, 1999); (2) the mutation in the obese patients as CYS105THR (Licinio et al, 2004); (3) an argl05-to-trp amino acid replacement as was noted in obese patients (Strobel et al, 1998); (4) homozygosity for the delta- 133G mutation in leptin found in obese patients (for the guanine deletion) (Gibson et al, 2004; Farooqi et al. 2002); (5) a deletion of a guanine nucleotide in the leptin gene, with 5 rather than the expected 6 guanine nucleotides present between nucleotides 393 and 398, where the mutation disrupts the reading frame of the leptin gene, leading to the introduction of 14 aberrant amino acids after glyl32 in the leptin polypeptide, followed by a premature stop codon (Montague et al, 1997); (6) a 538C/T polymorphism (Gaukrodger et al, 2005); (7) an arg 128-to-gln (Rl 28Q) substitution that does not affect receptor binding but knocks out biologic activity (Verpleogen et al, 1997); (8) frameshift mutation resulting in deletion of a single guanine nucleotide in codon 133 of the leptin gene (Montague et al, 1997); (8) a 144G-A transition in codon 48 and a 328G-A transition in codon 110 (Karvonen et al , 1998).

A variant polypeptide can have any degree of amino acid identify with a wild-type polypeptide. For example, a variant polypeptide may have at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or greater amino acid identity with the corresponding wild-type polypeptide. In some embodiments, the variant polypeptide is a polypeptide comprising at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, or more consecutive amino acids of SEQ ID NO:1. In some embodiments, the variant polypeptide is a polypeptide comprising at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, or more consecutive amino acids of SEQ ID NO: 105. In some embodiments, the variant polypeptide is a polypeptide comprising at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, or more consecutive amino acids of SEQ ID NO:14 or SEQ ID NO:15.

The present invention may utilize polypeptides purified from a natural source or from recombinantly-produced material. Those of ordinary skill in the art would know how to produce these polypeptides from recombinantly-produced material. This material may use the 20 common amino acids in naturally synthesized proteins, or one or more modified or unusual amino acids. Generally, "purified" will refer to a composition comprising a leptin polypeptide or variant leptin polypeptide that has been subjected to fractionation to remove various other proteins, polypeptides, or peptides, and which composition substantially retains its activity. Purification may be substantial, in which the leptin polypeptide or variant leptin polypeptide is the predominant species, or to homogeneity, which purification level would permit accurate degradative sequencing.

Amino acid sequence mutants of leptin polypeptides also are encompassed as "variant leptin polypeptides." Amino acid sequence mutants of the polypeptide can be substitutional mutants or insertional mutants. Insertional mutants typically involve the addition of material at a non-terminal point in the peptide. This may include the insertion of a few residues; an immunoreactive epitope; or simply a single residue. The added material may be modified, such as by methylation, acetylation, and the like. Alternatively, additional residues may be added to the N-terminal or C-terminal ends of the peptide. Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, or example, their hydrophobicity, hydrophilicity, charge, size, and the like. An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all a similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents.

Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, or example, their hydrophobicity, hydrophilicity, charge, size, and the like. An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all a similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents.

In making changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (- 0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporated by reference herein). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within + 2 is preferred, those which are within +1 are particularly preferred, and those within + 0.5 are even more particularly preferred.

It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent protein. As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 + 1); glutamate (+3.0 + 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 + 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within + 2 is preferred, those which are within + 1 are particularly preferred, and those within + 0.5 are even more particularly preferred.

Certain embodiments of the present invention utilize fusion proteins that are preferentially translocated through biological membranes. In particular, the leptin polypeptide or leptin polypeptide equivalent may be fused to a particular protein, polypeptide, or peptide sequence that promotes facilitated intracellular delivery of the fusion protein into the targeted cell. Although any fusion protein with the property of facilitated intracellular delivery is contemplated by the present invention, specific examples include fusion proteins utilizing the HIV TAT sequence (Nagahara et al., 1998), the third helix of the Antennapedia homeodomain (Antp) (Derossi et al, 1994), and the HSV-I structural protein VP22 (Elliott and O'Hare, 1997).

Examples of variant leptins known in the art include SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, MVPIQK (SEQ ID NO:27), TLIK (SEQ ID NO:28), TIVTR (SEQ ID NO:29), INDISHTQSVSSK (SEQ ID NO:30), VTGLDFIPGLHPILTLSK (SEQ ID NO:31), NVIQISNDLENLR (SEQ ID NO:32), DLLHVLAFSK (SEQ ID NO:33), SCHLPWASGLETLDSLGGVLEASGYSTEVVALSR (SEQ ID NO:34), LQGSLQDML WQLDLSPGC (SEQ ID NO:35), and VTGLDFIPGLHPILTLSK (SEQ ID NO:36). Other examples of leptin polypeptides and variant leptin polypeptides include those sequences set forth in WO96/05309, WO 96/40912, WO 97/06816, WO 00/20872, WO 97/18833, WO 97/38014, WO 98/08512, and WO 98/28427. Additional examples are set forth in U.S. Pat. Nos. 5,521,283; 5,525,705; 5,532,336; 5,552,522; 5,552,523; 5,552,524; 5,554,727; 5,559,208; 5,563,243; 5,563,244; 5,563,245; 5,567,678; 5,567,803; 5,569,743; 5,569,744; 5,574,133; 5,580,954; 5,594,101; 5,594,104; 5,605,886; 5,614,379; 5,691,309; 5,719,266, U.S. Patent App. Pub. No. 20050106679, each of which is herein specifically incorporated by reference in its entirety. Further examples are set forth in WO96/23513; WO96/23514; WO96/23515; WO96/23516; WO96/23517; WO96/23518; WO96/23519; WO96/34111; WO 96 37517; WO96/27385; WP 97/00886; EP 725078; EP 725079; EP 744408; EP 745610; EP 835879, WO96/22308, WO96/31526, WO96/34885; WO 97/46585, WO 96/35787, WO97/16550, WO 97/20933, EP 736599, and EP 741187. Each of the foregoing references in this paragraph is herein specifically incorporated by reference.

Additional examples of variant leptin polypeptides include those variants of the wild- type mouse leptin sequence set forth in Peelman et al, 2004, herein specifically incorporated by reference. These mutants include D95T12Q, L13N, K155, T16N, R20N, Q28S, S29QV30QS31N, A32N, Q34SR35S, F41S, H46A, T66A, Q75S, N78S, N825D855, L86A, L89A, F92A, S97Q, QlOOS, Q105S, Q108S, E108S, Dl I lS, E115S, E115S, S117Q, L118Q, S120AT121A, E122S, Q134S, D135S, Q138SQ139SV142A. Other examples of mutant L39A/D40A/F41A, mutant L39A/D40A/F41A/I42A, mutant L39A/D40A/F41A, mutant L39A/D40A/F41A/I42A, mutant L39A/D40A/F41A, and mutant L39A/D40A/F41A.

B. Polynucleotides Encoding a Leptin Polypeptide, a Leptin Transporter Polypeptide, a Leptin Receptor Polypeptide, or Variant Thereof

In certain embodiments of the present invention, the method of treatment or prevention of Parkinson's disease involves administering a pharmaceutically effective amount of a leptin polypeptide, leptin transporter polypeptide, leptin receptor polypeptide, or variant thereof, wherein administering involves contacting the subject with a polynucleotide encoding leptin polypeptide, leptin transporter polypeptide, leptin receptor polypeptide, or a variant thereof.

The polynucleotide encoding the full length amino acid sequence of human leptin is provided herein as SEQ ID NO:37. Additional examples of polynucleotides encoding a leptin polypeptide, leptin receptor polypeptide, leptin transporter polypeptide, or variant thereof are set forth in SEQ ID NOs 38- 103.

The polynucleotides according to the present invention may encode a full-length leptin protein sequence, a full-length leptin receptor sequence, a full-length leptin transporter sequence, or less than a full-length protein sequence. Further, the polynucleotide may include additional nucleic acid sequences that do not encode a leptin polypeptide, a leptin receptor polypeptide, a leptin transporter polypeptide, or variant of a leptin polypeptide. The polynucleotides may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism.

In other embodiments, however, the polynucleotides may be complementary DNA

(cDNA). cDNA is DNA prepared using messenger RNA (mRNA) as a template. Thus, a cDNA does not contain any interrupted coding sequences and usually contains almost exclusively the coding region(s) for the corresponding protein. In other embodiments, the polynucleotide may be produced synthetically.

It may be advantageous to combine portions of the genomic DNA with cDNA or synthetic sequences to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone will need to be used. Introns may be derived from other genes in addition to leptin. The cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.

The polynucleotides encoding leptin polypeptides may be naturally-occuring homologous polynucleotide sequences from other organisms. A person of ordinary skill in the art would understand that commonly available experimental techniques can be used to identify or synthesize polynucleotides encoding leptins or variants of leptins. The present invention also encompasses chemically synthesized mutants of these sequences.

Another kind of sequence variant results from codon variation. Because there are several codons for most of the 20 normal amino acids, many different DNAs can encode the Spi2A. Reference to the following table will allow such variants to be identified.

TABLE 1

Amino Acids Codons

Alanine Ala A GCA GCC GCG GCU

Cysteine Cys C UGC UGU

Aspartic acid Asp D GAC GAU

Glutamic acid GIu E GAA GAG

Phenylalanine Phe F UUC UUU

Glycine GIy G GGA GGC GGG GGU

Histidine His H CAC CAU

Isoleucine He I AUA AUC AUU

Lysine Lys K AAA AAG

Leucine Leu L UUA UUG CUA CUC CUG CUU

Methionine Met M AUG

Asparagine Asn N AAC AAU

Proline Pro P CCA CCC CCG CCU

Glutamine GIn Q CAA CAG

Arginine Arg R AGA AGG CGA CGC CGG CGU

Serine Ser S AGC AGU UCA UCC UCG UCU

Threonine Thr T ACA ACC ACG ACU

Valine VaI V GUA GUC GUG GUU

Tryptophan Trp W UGG

Tyrosine Tyr Y UAC UAU

Allowing for the degeneracy of the genetic code, sequences that have between about 50% and about 75%, or between about 76% and about 99% of nucleotides that are identical to the nucleotides disclosed herein will be preferred. Sequences that are within the scope the polynucleotides used in the methods set forth herein are those that are capable of base-pairing with a polynucleotide segment set forth above under intracellular conditions. As stated above, the polynucleotides employed in the methods set forth herein may be full length genomic or cDNA copies, or large fragments thereof. The present invention also may employ shorter oligonucleotides. Sequences of 12 bases long should occur only once in the human genome and, therefore, suffice to specify a unique target sequence. In certain embodiments, one may wish to employ constructs which include other elements, for example, those which include C-5 propyne pyrimidines. Oligonucleotides which contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity (Wagner et al, 1993).

C. Treatment of Disease 1. Definitions

The methods set forth herein pertain to the treatment or prevention of Parkinson's disease. As used, herein, "Parkinson's disease" (also known as paralysis agitans, shaking palsy, primary parkinsonism, or idiopathic Parkinson's disease) is a slowly progressive neurological disease. The four primary symptoms are tremor or trembling in hands, arms, legs, jaw, and face; rigidity or stiffness of the limbs and trunk; bradykinesia or slowness of movement; and postural instability or impaired balance and coordination. As these symptoms become more pronounced, patients may have difficulty walking, talking, or completing other simple tasks. Diagnosis of Parkinson's disease can be by any method known to those of ordinary skill in the art, such as neurological examination.

Since leptin treatment can reverse the neurotoxins-induced dopaminergic neuronal degeneration, this invention has applicability in the treatment of other forms of parkinsonism (secondary parkinsonism). These include: tumors in the brain; repeated head trauma; drug- induced parkinsonism - prolonged use of tranquilizing drugs, such as the phenothiazines, butyrophenones, reserpine, and the commonly used drug, metoclopramide for stomach upset; antipsychotics-induced parkinsonism; toxin-induced parkinsonism - manganese and carbon monoxide poisoning; postencephalitic parkinsonism - a viral disease that causes "sleeping sickness."; striatonigral degeneration - the substantia nigra of the brain is only mildly affected, while other areas of the brain show more severe damage; parkinsonism that accompanies other neurological conditions - such as Shy-Drager syndrome (multiple system atrophy), progressive supranuclear palsy, Wilson's disease, Huntington's disease, Hallervorden-Spatz syndrome, Alzheimer's disease, Creutzfeldt-Jakob disease, olivopontocerebellar atrophy, and post-traumatic encephalopathy. "Treatment" and "treating" as used herein refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition. For example, in the context of the present invention, treatment includes administration of a pharmaceutically effective amount of a leptin or variant thereof that is sufficient to result in lessening of the severity of any of the symptoms associated with Parkinson's disease or secondary parkinsonism.

The term "therapeutic benefit" or "therapeutically effective" as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, regarding the treatment of Parkinson's disease, a therapeutic benefit is obtained when the motor symptoms associated with the disease decrease in severity.

"Prevention" and "preventing" are used according to their ordinary and plain meaning to mean "acting before" or such an act. In the context of a particular disease or health-related condition, those terms refer to administration or application of an agent, drug, or remedy to a subject or performance of a procedure or modality on a subject for the purpose of blocking the onset of a disease or health-related condition. For example, a subject with no signs or symptoms of Parkinson's disease but who has a strong family history of Parkinson's disease may be administered a leptin or varient thereof for the purpose of blocking the onset of any of the signs or symptoms of Parkinson's disease. For example, the subject may be a subject carrying a gene that places the subject at risk for the development of Parkinson's disease. These genes include PARKl, PARK2, PARK3, PARK4, PARK5, PARK6, PARK7, PARK8, and PARK9. These, in some embodiments, the subject is a subject that is known or suspected to place the subject at risk of the development of Parkinson's disease. In such embodiments, repeat therapy over a prolonged duration of time may be required.

As used herein, treatment of Parkinson's disease or secondary parkinsonism includes any or all of the following: improvement in the severity of signs or symptoms associated with Parkinson's disease; resolution of any of the signs or symptoms associated with Parkinson's disease, reduction in the rate of dopaminergic neuronal cell degeneration in a subject with Parkinson's disease.

In additional embodiments of the invention, methods include identifying a patient in need of treatment or prevention of Parkinson's disease or secondary parkinsonism. A patient may be identified, for example, based on taking a patient history, or based on findings on clinical examination.

D. Gene Transfer In those embodiments of the present methods wherein a polynucleotide is administered to the subject, gene transfer into cells of the subject is desired. Gene transfer can be by any method known to those of ordinary skill in the art. For example, gene transfer may be through the use of a viral vector or a nonviral vector. The state of the art pertaining to gene transfer techniques is advanced, and any technique known to those of ordinary skill in the art is contemplated for application in the context of the present invention. The following sets forth examples of some vectors that can be applied in the context of the present invention. Information regarding gene transfer into neurons or gene therapy in the brain can be found in the following references, each of which is specifically incorporated by reference: Mochizuki and Mizuno, 2003; Mandel and Burger, 2004; Bjorklund et al, 2000; Glorioso et al, 2003; Kirik and Bjorklund, 2003; Ehrengruber, 2002; Washbourne and McAllister, 2002; Glorioso and Fink, 2004; Tenenbaum et al, 2004; Hendriks et al, 2004; and Suhr and Gage, 1999.

1. Viral Vectors

In certain embodiments, the methods of the invention utilize expression cassette which includes a polynucleotide encoding a leptin polypeptide, leptin transporter polypeptide, leptin receptor polypeptide, or variant thereof in an expression cassette carried in a viral vector. The term "expression cassette" is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. In order for the expression cassette to effect expression of a polypeptide, the polynucleotide encoding the polynucleotide will be under the transcriptional control of a promoter. A "promoter" is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrase "operatively linked" mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence. Techniques using "viral vectors" are well-known in the art. A viral vector is meant to include those constructs containing viral sequences sufficient to (a) support packaging of the expression cassette and (b) to ultimately express a recombinant gene construct that has been cloned therein. One method for delivery of the recombinant DNA involves the use of an adenovirus expression vector. Although adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors.

Adenoviruses are currently the most commonly used vector for gene transfer in clinical settings. Among the advantages of these viruses is that they are efficient at gene delivery to both nondividing and dividing cells and can be produced in large quantities. The vector comprises a genetically engineered form of adenovirus. Knowledge of the genetic organization or adenovirus, a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus et al, 1992). In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification.

Adenovirus is particularly suitable for use as a gene transfer vector because of its mid- sized genome, ease of manipulation, high titer, wide target-cell range and high infectivity. A person of ordinary skill in the art would be familiar with experimental methods using adenoviral vectors.

The adenovirus vector may be replication defective, or at least conditionally defective, and the nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector. Adenovirus growth and manipulation is known to those of skill in the art, and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 10⁹-10^π plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al., 1963; Top et al, 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors. Other viral vectors that can be applied in the methods set forth herein include retroviruses, adeno- associated viruses (AAV), herpes simplex virus (HSV), vaccinia virus vectors, and Venezuelan equine encephalitis (VEE) virus.

AAV vectors has been widely used clinically for gene transfer due to non- pathogenicity (most people carry this harmless virus), non-immunogenicity, induction of long-term gene transfer, and an ability to infect dividing and non-dividing cells such as neurons. Clinical trials have been initiated where AAV are used to deliver genes to the brain (see, e.g., Mandel and Burger, 2004).

A polynucleotide may be housed within a viral vector that has been engineered to express a specific binding ligand. The virus particle will thus bind specifically to the cognate receptors of the target cell and deliver the contents to the cell. A novel approach designed to allow specific targeting of retrovirus vectors was developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification can permit the specific infection of hepatocytes via sialoglycoprotein receptors.

Another approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used. The antibodies were coupled via the biotin components by using streptavidin (Roux et al, 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro (Roux et al., 1989).

2. Nonviral Vectors

Several non-viral methods for the transfer of expression vectors into cells also are contemplated by the present invention. These include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE- dextran (Gopal, 1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al., 1979) and liofectamine-DNA complexe, cell sonication (Fechheimer et al., 1987), gene bombardment using high velocity microprojectiles (Yang et al., 1990), polycations (Bousssif et al., 1995) and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use. A person of ordinary skill in the art would be familiar with the techniques pertaining to use of nonviral vectors, and would understand that other types of nonviral vectors than those disclosed herein are contemplated by the present invention. In a further embodiment of the invention, the expression cassette may be entrapped in a liposome or lipid formulation. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. One of ordinary skill in the art would be familiar with techniques utilizing liposomes and lipid formulations.

E. Pharmaceutical Preparations

Certain of the methods set forth herein pertain to methods involving the administration of a pharmaceutically effective amount of a leptin or variant thereof, or a pharmaceutically effective amount of a composition comprising a leptin or variant thereof. The phrases "pharmaceutically effective amount," "pharmaceutically acceptable," and "pharmaceutically effective" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate. 1. Compositions Comprising Leptins

In some embodiments of the present invention, a composition comprising a leptin or variant thereof and one or more pharmaceutically acceptable carriers is administered to a subject. Such compositions can be prepared using any method known to those of ordinary skill in the art, and can include any ingredient known to those of ordinary skill in the art. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives {e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (Remington's, 1990). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated. The compositions used in the present invention may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions, and these are discussed in greater detail below. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

The leptin polypeptide or variant thereof may be extensively dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle, where appropriate. The active compounds will then generally be formulated for administration by any known route, such as parenteral administration. Methods of administration are discussed in greater detail below.

In certain embodiments, the composition comprising a leptin polypeptide or variant thereof is an aqueous composition. The preparation of an aqueous composition containing an active agent of the invention disclosed herein will be known to those of skill in the art in light of the present disclosure. Aqueous compositions of the present invention comprise an effective amount an of a leptin polypeptide or variant dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

In some embodiments, the leptin polypeptide or variant is formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. A person of ordinary skill in the art would be familiar with techniques for generation of salt forms. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.

The present invention contemplates methods using compositions that are sterile solutions for intravascular injection or for application by any other route as discussed in greater detail below. A person of ordinary skill in the art would be familiar with techniques for generating sterile solutions for injection or application by any other route. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients familiar to a person of skill in the art. The formulation of the composition may vary depending upon the route of administration. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.

In addition to the compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; liposomal and nanoparticle formulations; time release capsules; formulations for administration via an implantable drug delivery device, and any other form. One may also use nasal solutions or sprays, aerosols or inhalants in the present invention.

Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. A person of ordinary skill in the art would be familiar with well-known techniques for preparation of oral formulations.

In certain embodiments, pharmaceutical composition includes at least about 0.1% by weight of the active compound. In other embodiments, the pharmaceutical composition includes about 2% to about 75% of the weight of the composition, or between about 25% to about 60% by weight of the composition, for example, and any range derivable therein.

The pharmaceutical composition may comprise various antioxidants to retard oxidation of one or more component. For example, the composition may include a peptidase inhibitor. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof. The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that exotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.

The leptin polypeptide or variant may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.

In other embodiments, one may use nasal solutions or sprays, aerosols or inhalants in the present invention. Nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays.

Solid compositions for oral administration are also contemplated by the present invention. In these embodiments, the solid composition may comprise, for example, solutions, suspensions, emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatin capsules), sustained release formulations, buccal compositions, troches, elixirs, suspensions, syrups, wafers, or combinations thereof. Oral compositions may be incorporated directly with the food of the diet. Preferred carriers for oral administration comprise inert diluents, assimilable edible carriers or combinations thereof. In other aspects of the invention, the oral composition may be prepared as a syrup or elixir. A syrup or elixir, and may comprise, for example, at least one active agent, a sweetening agent, a preservative, a flavoring agent, a dye, a preservative, or combinations thereof.

In certain preferred embodiments an oral composition may comprise one or more binders, excipients, disintegration agents, lubricants, flavoring agents, and combinations thereof. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. Additional formulations which are suitable for other modes of administration include suppositories. Suppositories are solid dosage forms of various weights and shapes for insertion into the rectum, vagina or urethra.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.

In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof. 2. Routes of Administration

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.

The composition comprising a leptin polypeptide or variant thereof can be administered to the subject using any method known to those of ordinary skill in the art. For example, a pharmaceutically effective amount of the composition may be administered intravenously, intracerebrally, intracranially, intrathecally, into the substantia nigra or the region of the substantia nigra, intradermally, intraarterially, intraperitoneally, intralesionally, intratracheally, intranasally, topically, intramuscularly, intraperitoneally, subcutaneously, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (Remington's, 1990). Administration may be continuous (such as by infusion through a minipump or catheter) or intermittent (such as by injections). In particular embodiments, the composition comprising a leptin polypeptide or variant thereof is administered to a subject using a drug delivery device. Any drug delivery device is contemplated for use in delivering a pharmaceutically effective amount of a composition comprising a leptin polypeptide or variant thereof as set forth herein. For example, in particular embodiments the drug delivery device is an osmotic mini-pump. Other examples of drug delivery devices contemplated for use in the methods of the present invention include nasal drug delivery; oral drug delivery, topical drug delievery (such as tranbuccal, transmucosal and transdermal delivery, subcutaneous drug delivery), intraperitoneal injection, intramuscular injection, intravascular drug delivery, intravenous infusion, subcutaneous injection or infusion, epidural drug delivery, intradural drug delivery, intraventricular drug delivery, intracranial administration, intraethecal administration, and direct drug delivery to specific brain regions especially the substantia nigra and caudate-putamen(striatum). 3. Dosage A pharmaceutically effective amount of the leptin polypeptide or variant thereof is determined based on the intended goal, for example inhibition of cell death. The quantity to be administered, both according to number of treatments and dose, depends on the subject to be treated, the state of the subject, the protection desired, and the route of administration. Precise amounts of the therapeutic agent also depend on the judgment of the practitioner and are peculiar to each individual. For example, a dose of the leptin polypeptide or variant thereof may be about 0.0001 milligrams to about 1.0 milligrams, or about 0.001 milligrams to about 0.1 milligrams, or about 0.1 milligrams to about 1.0 milligrams, or even about 10 milligrams per dose or so. Multiple doses can also be administered. In some embodiments, a dose is at least about 0.0001 milligrams. In further embodiments, a dose is at least about 0.001 milligrams. In still further embodiments, a dose is at least 0.01 milligrams. In still further embodiments, a dose is at least about 0.1 milligrams. In more particular embodiments, a dose may be at least 1.0 milligrams. In even more particular embodiments, a dose may be at least 10 milligrams. In further embodiments, a dose is at least 100 milligrams or higher.

In other non-limiting examples, a dose may also comprise from about 0.1 microgram/kg/body weight, about 0.2 microgram/kg/body weight, about 0.3 microgram/kg/body weight, about 0.4 microgram/kg/body weight, about 0.5 microgram/kg/body weight, about 0.6 microgram/kg/body weight, about 0.7 microgram/kg/body weight, about 0.8 microgram/kg/body weight, about 0.9 microgram/kg/body weight, about 1.0 microgram/kg/body weight, about 2.0 microgram/kg/body weight, about 3.0 microgram/kg/body weight, about 4.0 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non- limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.

The dose can be repeated as needed as determined by those of ordinary skill in the art. Thus, in some embodiments of the methods set forth herein, a single dose of the leptin polypeptide or variant thereof is contemplated. In other embodiments, two or more doses of the leptin polypeptide or variant thereof are contemplated. Where more than one dose is administered to a subject, the time interval between doses can be any time interval as determined by those of ordinary skill in the art. For example, the time interval between doses may be about 1 hour to about 2 hours, about 2 hours to about 6 hours, about 6 hours to about 10 hours, about 10 hours to about 24 hours, about 1 day to about 2 days, about 1 week to about 2 weeks, or longer, or any time interval derivable within any of these recited ranges.

In certain embodiments, it may be desirable to provide a continuous supply of a pharmaceutical composition to the patient. For example, a patient with Parkinson's disease may be treated with a continuous intravascular administration of the therapeutic agent for a defined period of time. Continuous perfusion of the region of interest (such as the region of the substantia nigra) is contemplated in some embodiments. This could be accomplished by catheterization, followed by continuous administration of the therapeutic agent. The administration could be intra-operative or post-operative. 4. Secondary Treatment

Certain embodiments of the present invention provide for the administration or application of one or more secondary forms of therapies for the treatment or prevention of Parkinson's disease. The secondary form of therapy may be administration of one or more secondary pharmacological agents that can be applied in the treatment or prevention of Parkinson's disease. Examples of such agents include levodopa, a dopamine agonist, selegiline, an anticholinergic agent, amantadine, a catechol O-methyltransferase (COMT) inhibitor, an anti- inflammatory agent, an antioxidant, an antiapoptotic agent, or a neurotrophic agent.

Other secondary therapies include surgery, such as ablation procedures, deep brain stimulation, pallidotomy, or cerebral transplantation (e.g., implantation of dopamine- producing cells into the brain). Also contemplated as secondary therapies are physical therapy and nutritional supplements. If the secondary therapy is a pharmacological agent, it may be administered prior to, concurrently, or following administration of the leptin polypeptide or variant thereof. In some embodiments, the secondary agent is included as an additional ingredient in a composition comprising a leptin polypeptide or variant thereof as discussed above.

In further embodiments, the secondary therapeutic agent is included in a composition that is separate from the composition comprising the leptin polypeptide or variant thereof. These compositions would be provided in a combined amount to treat or prevent Parkinson's disease.

The interval between the leptin polypeptide or variant thereof and the secondary therapy may be any interval as determined by those of ordinary skill in the art. For example, the interval may be minutes to weeks. In embodiments where the agents are separately administered, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that each therapeutic agent would still be able to exert an advantageously combined effect on the subject. For example, the interval between therapeutic agents may be about 12 h to about 24 h of each other and, more preferably, within about 6 hours to about 12 h of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several d (1, 2, 3, 4, 5, 6 or 7) to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations. In some embodiments, the timing of administration of a secondary therapeutic agent is determined based on the response of the subject to the leptin or variant thereof.

F. Kits

Certain embodiments of the present invention are generally concerned with kits comprising at least one sealed vial and a leptin polypeptide or variant thereof. For example, the kit may include at least one sealed vial that includes a leptin polypeptide or variant thereof. In some embodiments, the kit includes 2 or more sealed vials, at least one of which includes a leptin polypeptide or variant thereof. In some embodiments, the kit includes one or more additional therapeutic agents that can be applied in the treatment or prevention of Parkinson's disease. Examples of such additional therapeutic agents are discussed elsewhere in this specification.

The kit may also contain conventional pharmaceutical carriers and adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, buffers, preservatives, antioxidants, surfactants, and the like.

The components of the kit may be in liquid, frozen or dry form. The sealed container may or may not be a container that is fabricated to protect the contents therein from exposure to light. The kit may further include instructions for administration of the composition and treatment of a subject in a manner as set forth herein. In some embodiments, the kit includes a catheter which can be applied in delivering the leptin or variant thereof to a subject. In some embodiments, the kit includes one or more drug delivery devices. Examples of drug delivery devices contemplated for inclusion in kits of the present invention are discussed elsewhere in this specification.

G. Methods of Screening

The present invention contemplates methods for screening and/or manufacturing agents that can be applied in the treatment or prevention of Parkinson's disease. These methods may comprise screening candidate substances for leptin activity or structural similarity to a leptin in accordance with any method known to those of ordinary skill in the art.

As used herein, the term "candidate substance" refers to any molecule that may potentially have leptin activity and/or structural similarity to a leptin. Candidate compounds may include fragments or parts of naturally-occurring compounds or may be found as active combinations of known compounds which are otherwise inactive. The candidate substance can be a nucleic acid, a polypeptide, a small molecule, etc.

One basic approach to search for a candidate substance is screening of compound libraries. One may simply acquire, from various commercial sources, small molecule libraries that are believed to meet the basic criteria for useful drugs in an effort to "brute force" the identification of useful compounds. Screening of such libraries, including combinatorially generated libraries, is a rapid and efficient way to screen a large number of related (and unrelated) compounds for activity. Combinatorial approaches also lend themselves to rapid evolution of potential drugs by the creation of second, third and fourth generation compounds modeled of active, but otherwise undesirable compounds. Screening assays may be in vivo, in vitro, or in cyto screening assays. For example, screening assays may involve assessing candidate substances for ability to protect against neurodeneration. For example, screening assays can be performed: (1) to measure whether a candidate substance increases leptin levels in blood, cerebrospinal fluid, brain tissue especially in the areas emcompassing the substantia nigra and caudate-putamen (striatum); leptin levels can be measured using radioimmunoassay (RIA) kits or ELISA methods; (2) to assess leptin activity by determining leptin receptor affinity for leptin using receptor binding assay with radiolabeled leptin; (3) to assess leptin activity of a candidate substance using a luciferase- based reporter assay (this will involve the use of cells that are transfected with leptin receptors, a luciferase reporter system is employed to quantify the leptin-mediated signals); and (4) to assess leptin activity of a candidate substance by measuring its ability to induce cell proliferation (for example, leptin activity can be determined by measuring proliferation of leptin-dependent human leptin receptor-transfected murine BaF3 cells).

Screening assays can also be performed to assess leptin activity of a candidate substance by measuring activity of leptin receptor-induced signal transduction. This includes the measurements of activation of signal transducer and activator of transcription (STAT), the serine/threonine protein kinase Akt/Protein kinase B, mitogen-activated protein kinase (MAPK)/extracelular signal-regulated kinase (ERK), AMP-activated protein kinase (AMPK); the mammalian target of rapomycin (mTOR), S6 kinase 1 (S6K1) and S6 ribosomal protein (S6). The activation of aforementioned proteins can be assessed in vitro and in vivo by measuring their phosphorylation forms using Western blot assays with specific antibodies;

It will be understood that an undesirable compound includes compounds that are typically toxic, but have been modified to reduce the toxicity or compounds that typically have little effect with minimal toxicity and are used in combination with another compound to produce the desired effect.

Thus, the present invention can perform random screening of large libraries of candidate substances; alternatively, the methods may be used to focus on particular classes of compounds selected with the aim of finding structural attributes that are believed to make them more likely to have therapeutic efficacy and selectivity.

Treatment of animals with test compounds involves the administration of the compound, in an appropriate form, to the animal. Administration is by any route that could be utilized for clinical or non-clinical purposes. Specifically contemplated are intracerebral, subcutaneous, nasal, intraperitoneal and intravenous administration.

It will, of course, be understood that all the screening methods of the present invention are useful in themselves notwithstanding the fact that effective candidates may not be found. The invention provides methods for screening for such candidates, not solely methods of finding them.

In an extension of any of the previously described screening assays, the present invention also provide for methods of producing or manufacturing pharmaceutical agents that can be applied in the treatment or prevention or Parkinson's disease. Manufacturing can entail any well known and standard technique used by those of skill in the art, such as synthesizing the compound and/or deriving the compound from a natural source.

H. Examples

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1 Dopaminergic Neurodegeneration Occurs in Leptin-Deficient Mice (ob/ob) Brain tissues from 4-month old leptin-deficient (ob/ob) mice and wild type C57/BL6 mice, purchased from Jackson Laboratories, were used for immunohistochemical staining with an anti-tyrosine hydroxylase (TH) antibody. TH is a key enzyme for dopamine synthesis, which has been used as a marker for the dopamine neurons in the substantia nigra. The TH-positive neurons were compared at two different anatomical levels of the SN (FIG. 1, panel A-B).

A dramatic decrease in the number of TH positive neurons (dopaminergic neurons) was observed at both levels in the SN of the ob/ob mouse (FIG. 1). Interestingly, selective loss of TH neurons was observed in the substantia nigra pars compacta (SNc), but not in the ventral tegmental area (VTA) (FIG. 1), which resembles the pattern of neurodegeneration of the dopamine neurons in PD patients. In addition, the density of TH immunoreactive fibers are also less in the ob/ob mouse than that in the wild type control group. These results suggest that leptin may be a critical factor for the survival of dopamine neurons, especially for the dopaminergic neurons in the SNc. EXAMPLE 2

Leptin Protects Dopaminergic Neurons in Primary Midbrain Cultures from MPP+ and

Rotenone

The protective effect of leptin on dopaminergic neurons was further determined using mouse primary midbrain (including the SN) neuronal cell cultures. The cells were pretreated with different concentrations of recominant rat leptin for 24 h and then challenged with 10 μM of l-methyl-4-phenylpyridinium species (MPP⁺), a metabolite of l-methyl-4-phenyl-5,6- dihydro-2-pyridone (MPTP), which is commonly used to induce Parkinson's disease in animal models. Leptin or MPP+ was applied to the culture medium. The rat recombinant leptin was purchased from R&D systems; the sequence of rat leptin is as set forth in SEQ ID NO:104.

The dopamine neurons were stained with a TH-antibody by Immunocytochemistry. The numbers of the TH-positive neurons were used as a measurement for leptin mediated protection of dopaminergic neurons from l-methyl-4-phenylpyridinium (MPP⁺). FIG. 2 shows the effect of leptin on the survival of TH-immunoreactive neurons in mouse primary mesencephalon cultures challenged with MPP+. 10 μM MPP+ induced a significant reduction in TH-immunoreactive (TH-ir) cell number compared to untreated control cells (*p<0.01). Leptin treatment in primary cultures protected dopaminergic neurons from MPP+ - induced cell death. Leptin, at all concentrations tested (0.04 μM, 0.2 μM and 1 μM), was able to fully protect the TH-ir cells from 10 μM MPP+-induced degeneration (**, p<0.01 compared to cells treated with 10 μM MPP+ and without leptin) (FIG. 3).

In the next experiments, studies were conducted to determine whether leptin is able to protect primary dopamine neurons from rotenone. Rotenone, a pesticide, is another neurotoxin that inhibits the mitochondria complex 1 activity (Degli Esposti, 1998). Rotenone has been used in animal models to generate Parkinsonism-like syndromes (Bove et ah, 2005; Soderstrom et ah, 2006). Primary cultured cells were pretreated with leptin (1 μM), and then the cells were challenged with two concentrations of rotenone (5 nM and 20 nM). The number of TH-positive neurons was counted. It showed that leptin was able to protect the cells from rotenone toxicity (FIG. 4). EXAMPLE 3 Leptin Protects Dopaminergic Neurons in Mice Treated With MPTP

Since leptin protects against MPP+-induced dopaminergic neuronal cell death in vitro, studies were conducted to test whether leptin is able to protect dopaminergic neurons in vivo using an MPTP-induced model of Parkinson's Disease. MPTP selectively destroys dopamine neurons in the substantia nigra and induces Parkinsonism syndromes in humans. The MPTP mouse model is a well-established PD model (Dauer and Przedborski, 2003). In the brain, MPTP is converted to MPP+ mainly by astroglia cells. MPP+ is released from astroglia cells and then taken up by dopamine neurons through the dopamine transporter (DAT) (Dauer and Przedborski, 2003). MPP+ poorly passes the blood-brain barrier; therefore, MPTP is used for in vivo studies. The animals used in these studies were 8 week-old C57BL/6J mice because of the large amount of data on the sensitivity of this strain of mice to MPTP treatment in this strain.

Leptin was administered using subcutaneously implanted osmotic minipumps (Alzet model 2002; Alza Corp., Palo Alto, CA). Osmotic minipumps delivered either saline or 2 mg/kg/24h leptin dissolved in saline for a total of 14 days at a rate of 0.5 μl/h. The food intake and body weight of the treated mice were recorded as an indication of the biological* effectiveness of leptin. As indicated in FIG. 5, body weight loss occurred one day after the minipump implantation surgery in mice treated with both leptin and saline. However, mice treated with leptin maintained low body weight significantly with reduced total food intake. In addition, we did not find any obvious side-effects other than slightly decreasing body weight in the leptin treated mice. Seven days after the start of leptin treatment, the treated mice were challenged with intraperitoneal MPTP at 20 mg/kg, 4 times at 2h intervals. Rotarod performance was tested on the animals at 2 and 7 days after MPTP challenge to evaluate motor function. FIG. 6 shows that MPTP caused a significant decrease in rotarod performance in mice pretreated with saline at day 2 after MPTP challenge. The decrease in rotarod performance in these mice resolves 7 days after MPTP treatment. Importantly, MPTP did not cause rotarod performance decrease in mice pretreated with leptin (* p<0.05, compared to the saline group challenged with MPTP), indicating that leptin was able to functionally protect mice from MPTP-induced motor impairments.

To determine whether leptin was able to protect the dopaminergic neuronal damage caused by MPTP, the treated animals were perfused with 4% paraformaldehyde at day 7 after MPTP challenge, and the brains were sectioned for immunohistochemistry with an anti-TH antibody. As shown in FIG. 7, MPTP caused a significant loss (90%) of TH-fibers in the striatum compared to the saline-treated control group (** p<0.001). When mice were pretreated with leptin, MPTP caused significantly less TH-fiber loss in the striatum compared to the saline pretreated group (* p<0.05). These results indicated that leptin is able to protect the dopaminergic neuronal fibers in the striatum from MPTP-induced degeneration.

EXAMPLE 4

Clinical Trials of the Use of Leptin Polypeptides, Leptin Transporter Polypeptides, Leptin Receptor Polypeptides, and Variants Thereof in Treating or Preventing Parkinson's Disease

This example is concerned with the development of human treatment protocols for the treatment and prevention of Parkinson's disease using the leptin polypeptides, leptin receptor polypeptides, leptin transporter polypeptides, or variants thereof as discussed above. The various elements of conducting a clinical trial, including patient treatment and monitoring, will be known to those of skill in the art in light of the present disclosure. The following information can be used as a general guideline for use in the treatment or prevention of Parkinson's disease, alone or in combination with other drugs in clinical trials.

Patients with Parkinson's disease or patients at risk of developing Parkinson's disease can be chosen for clinical study. For example, patients with Parkinson's disease may be offered participation because they may have failed to respond to at least one course of conventional therapy.

In an exemplary clinical protocol, patients may undergo placement of a drug delivery catheter, such as an intracerebral catheter. In the same procedure, the leptin polypeptide or variant leptin polypeptide may be administered alone or in combination with other therapeutic drugs that are commonly used in the treatment of Parkinson's disease. The administration, for example, may be into the region of the substantia nigra.

The starting dose may be 0.01-0.08 mg/kg body weight. Three patients may be treated at each dose level in the absence of grade > 3 toxicity. Dose escalation may be done by 100% increments (0.5 mg, 1 mg, 2 mg, 4 mg) until drug related grade 2 toxicity is detected. Thereafter dose escalation may proceed by 25% increments. The administered dose may be fractionated equally into two infusions, separated by six hour intervals if combined with a second drug for any given patient.

Additional information regarding dosages can be found in the following references: (1) Ebihara et al, 2007 (The physiological replacement dose was estimated to be 0.02 mg/kg/d for men, 0.03 mg/kg/d for girls under 18 yr of age, and 0.04 mg/kg/d for women on the basis of information provided by Amgen); (2) Javor et al, 2002 (the usual replacement dose was 0.06-0.08 mg/kg/day for females and 0.04 mg/kg/day for males in an attempt to simulate the normal to high physiologic range.); (3) Matochik et al, 2005 (The subjects received daily sc injections of recombinant methionyl human leptin at low physiological doses, in the range of 0.01-0.04 mg/kg, for the duration of the study.); (4) Musso et al, 2005 (the physiologic replacement dose was estimated to be 0.03 mg/kg/d for females under 18 years and 0.04 mg/kg/d for adult females; for all males, the dose was 0.02 mg/kg.); (5) Chan, 2005 (subjects received a single dose of r-metHuLeptin (0.3 mg/kg) by sc injection at 0800 h once daily for 3 consecutive days); (6) McDuffϊe et al, 2004 (the physiological replacement dose was estimated to be 0.03-0.04 mg/kg body weight); (7) Welt et al, 2004 (the subjects self- administered r-metHuLeptin (0.08 mg per kilogram of body weight per day) subcutaneously for two to three months, with 40 percent of the daily dose given at 8 a.m. and 60 percent at 8 p.m. to mimic the normal diurnal variation in leptin levels.); (8) Licinio et al, 2004 (patients received r-metHuLeptin s.c, once daily in the evening (18:00-20:00) at low physiological replacement doses in the range of 0.01-0.04 mg/kg for 18 months.); (9) Moran et al, 2004 (the physiologic replacement dose was approximated to be 0.03 mg/kg/d for female children under 18, 0.04 mg/kg/d for adult females, and 0.02 mg/kg/d for all adult males.); (10) Chan et al, 2003 (clinical-quality r-metHuLeptin was administered as four subcutaneous injections per day at a dose ranging from 0.001-0.008 mg/kg per day, based on the subject's baseline leptin level, and were administered every 6 hours starting at 8:00 am on day 1 through day 3 with one last dose at 8:00 am on day 4; for the replacement-dose r-metHuLeptin admission, the daily dose of r-metHuLeptin was 0.04 mg/kg per day on day 1, 0.1 mg/kg per day on days 2 and 3, and one dose of 0.025 mg/kg at 8:00 am on the fourth day, with the total daily dose divided into four equal doses given every 6 hours starting at 8:00 am on day 1.); (11) Simha et al, 2003 (0.04 mg/kg/day for women and 0.03 mg/kg/day for men); (12) Oral et al, 2002 (0.03 mg/kg per day for females under 18 yr and 0.04 mg/kg per day for adult females); and (13) Oral et al, 2002 (0.03 mg per kilogram of body weight per day for girls under 18 years of age and 0.04 mg per kilogram per day for women). The leptin polypeptides or variant leptin polypeptides, and any other drug used in combination, may be administered over a short infusion time followed by removal of the catheter. The leptin polypeptides or variant leptin polypeptides may be administered by infusion, alone or in combination with the other therapeutic agents. The infusion given at any dose level will be dependent upon the toxicity achieved after each administration. Increasing doses of the leptin polypeptide or variant leptin polypeptide alone or in combination with another therapeutic drug will be administered to groups of patients until approximately 60% of patients show unacceptable toxicity in any category. Doses that are 2/3 of this value could be defined as the safe dose. Physical examination and baseline studies such as CT scans or MRIs may, of course, be performed before treatment and at intervals of about 3-4 wk later. Laboratory studies should include CBC, differential and platelet count, urinalysis, SMA- 12- 100 (liver and renal function tests), coagulation profile, and any other appropriate chemistry studies.

To monitor disease course and evaluate the response to therapy, patients can, for example, be examined prior to therapy and following therapy at appropriate intervals.

Clinical responses may be defined by any acceptable measure known to those of skill in the art. For example, assessment for improvement or worsening of neurological symptoms of Parkinson's disease can be performed.

The leptin polypeptides or variant leptin polypeptides, and any other drug used in combination, may be administered over a short infusion time followed by removal of the catheter. The leptin polypeptides or variant leptin polypeptides may be administered by infusion, alone or in combination with the other therapeutic agents. The infusion given at any dose level will be dependent upon the toxicity achieved after each administration. Increasing doses of the leptin polypeptide or variant leptin polypeptide alone or in combination with another therapeutic drug will be administered to groups of patients until approximately 60% of patients show unacceptable toxicity in any category. Doses that are 2/3 of this value could be defined as the safe dose.

Physical examination and baseline studies such as CT scans or MRIs may, of course, be performed before treatment and at intervals of about 3-4 wk later. Laboratory studies should include CBC, differential and platelet count, urinalysis, SMA- 12- 100 (liver and renal function tests), coagulation profile, and any other appropriate chemistry studies.

To monitor disease course and evaluate the response to therapy, patients can, for example, be examined prior to therapy and following therapy at appropriate intervals.

Clinical responses may be defined by any acceptable measure known to those of skill in the art. For example, assessment for improvement or worsening of neurological symptoms of Parkinson's disease can be performed. EXAMPLE 5

Intermittent Intraperitoneal Administration of Leptin Prevents the Dopamine Fiber Loss in the MPTP Mouse Model of Parkinson's Disease Intermittent intraperitoneal (i.p.) administration of leptin prevents the loss of dopaminergic fibers in the striatum induced by MPTP. Mice received i.p. injections with saline or leptin twice daily for 7 days prior to MPTP challenge. FIG. 8A shows immunostaining of tyrosine hydroxylase (TH) for dopaminergic fibers in the striatum. FIG. 8B shows quantitative data of TH-immunreactive fiber density in the striatum. MPTP significantly decreased the density of dopaminergic fibers in the striatum (FIG. 8B). The loss of dopaminergic fibers induced by MPTP can be prevented by pretreatment with different doses of leptin.

All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Claims

CLAIMED

1. A method of treating or preventing Parkinson's disease or secondary parkinsonism in a subject, comprising administering to the subject a pharmaceutically effective amount of a leptin polypeptide, a leptin transporter polypeptide, a leptin receptor polypeptide, a synthetic leptin receptor ligand, or a variant thereof, wherein administering the leptin polypeptide, leptin transporter polypeptide, leptin receptor polypeptide or variant thereof results in treatment or prevention of Parkinson's disease in the subject.

2. The method of claim 1 , wherein the subject is a human.

3. The method of claim 1, wherein the subject has Parkinson's disease, and wherein the method is a method for treating Parkinson's disease.

4. The method of claim 1, wherein the subject is at risk of developing Parkinson's disease and wherein the method is a method for preventing Parkinson's disease.

5. The method of claim 1, wherein administering the leptin polypeptide, leptin transporter polypeptide, leptin receptor polypeptide, synthetic leptin receptor ligand, or a variant thereof reduces the rate of neuronal degeneration in the subject.

6. The method of claim 5, wherein the neuronal degeneration is further defined as dopaminergic neuronal degeneration.

7. The method of claim 1, wherein administering the leptin polypeptide, leptin transporter polypeptide, leptin receptor polypeptide, synthetic leptin receptor ligand, or a variant thereof results in improvement of motor deficits in the subject.

8. The method of claim 1, wherein administering is further defined as intravenous administration, intracerebral administration, administration into the cerebrospinal fluid, intraspinal administration, epidural administration, intralesional administration, topical administration, intraarterial administration, oral administration, regional administration, local administration, systemic administration, intraperiotoneal administration, intratumoral administration, intrathecal administration, subcutaneous administration, or administration via an implantable delivery device.

9. The method of claim 1, wherein administering is further defined as intravenous administration.

10. The method of claim 9, wherein administering is further defined as intracerebral administration.

11. The method of claim 10, wherein administering is further defined as administration via an implantable delivery device.

12. The method of claim 11, wherein the implantable delivery device is further defined as a subcutaneously implanted osmotic minipump.

13. The method of claim 1, wherein the leptin polypeptide, leptin transporter polypeptide, leptin receptor polypeptide, synthetic leptin receptor ligand, or a variant thereof is provided to the subject by administering to the subject a nucleic acid sequence encoding a leptin polypeptide, leptin transporter polypeptide, leptin receptor polypeptide, synthetic leptin receptor ligand, or variant thereof.

14. The method of claim 13, wherein the nucleic acid is comprised within an expression construct, and wherein the nucleic acid sequence is under the transcriptional control of a promoter.

15. The method of claim 14, wherein the nucleic acid encodes a polypeptide that comprises at least 10 contiguous amino acids of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:20.

16. The method of claim 13, wherein the expression construct is a viral vector.

17. The method of claim 14, wherein the viral vector is an adenovirus vector, an adeno-associated virus vector, a herpesvirus vector, a retrovirus vector, a lentivirus vector, a vaccinia virus vector, or a polyoma vector.

18. The method of claim 17, wherein the viral vector is an adenovirus vector.

19. The method of claim 13, wherein the nucleic acid is comprised within a liposome.

20. The method of claim 1, further comprising identifying a subject in need of treatment or prevention of Parkinson's disease.

21. The method of claim 1, further comprising measuring a response to therapy following administration of the leptin polypeptide or variant thereof.

22. The method of claim 21, wherein measuring a response to therapy comprises assessment of motor function of the subject before and after administering the leptin polypeptide or variant thereof.

23. The method of claim 1, further comprising administering one or more secondary forms of therapy to the subject for the treatment or prevention of Parkinson's disease or secondary parkinsonism.

24. The method of claim 23, wherein the secondary form of therapy is selected from the group consisting of pharmacological therapy, gene therapy, immunotherapy, surgical therapy, cell transplantation therapy, herbal medicines or acupuncture, complementary therapies such as Yoga and Tai Chi, therapeutic massage, and dietary supplements..

25. The method of claim 24, wherein the secondary form of therapy is pharmacological therapy.

26. The method of claim 25, wherein the pharmacological therapy is levodopa, a dopamine agonist, selegiline, an anticholinergic, amantadine, a catechol 0-methyltransferase (COMT) inhibitor, an anti-inflammatory agent, an antioxidant, an anti-apoptotic agent, or a neurotrophic factor.

27. The method of claim 1, wherein the leptin polypeptide or variant thereof is administered at a dose of about 0.1 μg/kg/24 hr to about 1 g/kg/24hr.

28. The method of claim 27, wherein the leptin polypeptide or variant thereof is administered at a dose of about 0.1 μg/kg/24 hr to about 500 mg/kg/24 hr.

29. The method of claim 28, wherein the leptin polypeptide or variant thereof is administered at a dose of about 0.5 μg/kg/24 hr to about 50 mg/kg/24 hr.

30. The method of claim 29, wherein the leptin polypeptide or variant thereof is administered at a dose of about 0.2 mg/kg/24 hr to about 5 mg/kg/24 hr.

31. The method of claim 1 , comprising administering a leptin polypeptide.

32. The method of claim 31, wherein the leptin polypeptide comprises at least 50 contiguous amino acids of SEQ ID NO: 1.

32. The method of claim 31, wherein the leptin polypeptide comprises at least 100 contiguous amino acids of SEQ ID NO: 1.

33. The method of claim 32, wherein the leptin polypeptide comprises at least 500 contiguous amino acids of SEQ ID NO: 1.

34. The method of claim 1, comprising administering a variant of a leptin polypeptide.

35. The method of claim 34, wherein the variant of a leptin polypeptide comprises an amino acid sequence comprising SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36.

36. The method of claim 1, wherein administering comprises administering to the subject a pharmaceutically effective amount of a composition comprising a leptin polypeptide or variant thereof and a pharmaceutically acceptable carrier.

37. The method of claim 36, wherein the composition comprises a secondary therapeutic agent.

38. The method of claim 37, wherein the secondary therapeutic agent is levodopa, a dopamine agonist, selegiline, an anticholinergic, amantadine, a catechol O- methyltransferase (COMT) inhibitor, an anti-inflammatory agent, an antioxidant, an anti- apoptotic agent, or a neurotrophic factor.

39. A kit comprising at least one sealed vial, wherein the vial comprises a leptin polypeptide or variant thereof.

40. The kit of claim 39, further comprising a catheter or delivery device.