WO2012170452A2 - Compositions and methods for treating neurodegenerative diseases - Google Patents

Compositions and methods for treating neurodegenerative diseases Download PDF

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Publication number
WO2012170452A2
WO2012170452A2 PCT/US2012/040970 US2012040970W WO2012170452A2 WO 2012170452 A2 WO2012170452 A2 WO 2012170452A2 US 2012040970 W US2012040970 W US 2012040970W WO 2012170452 A2 WO2012170452 A2 WO 2012170452A2
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ngf
subject
amino acid
method
mutein
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PCT/US2012/040970
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French (fr)
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WO2012170452A3 (en
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Chengbiao Wu
William C. Mobley
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The Regents Of The University Of California
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Publication of WO2012170452A3 publication Critical patent/WO2012170452A3/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18Growth factors; Growth regulators
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475Growth factors; Growth regulators
    • C07K14/48Nerve growth factor [NGF]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18Growth factors; Growth regulators
    • A61K38/185Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3

Abstract

The invention provides methods and compositions for treating neurodegenerative disorders without pain associated with traditional treatments by administering a nerve growth factor (NGF) mutein. The invention also provides methods and compositions for treating pain in a subject.

Description

COMPOSITIONS AND METHODS FOR

TREATING NEURODEGENERATIVE DISEASES

GRANT INFORMATION

[0001] This invention was made with government support under Grant No. NS055371 , awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

[0002] The present invention relates generally to medicine and the treatment of neurodegenerative diseases and disorders, and more particularly, to compositions and methods for treating such disorders without eliciting pain associated with traditional treatments.

BACKGROUND INFORMATION

[0003] Pain has been defined in a variety of ways. For example, pain can be defined as the perception by a subject of noxious stimuli that produces a withdrawal reaction by the subject. The most commonly experienced form of pain may be defined as the effect of a stimulus on nerve endings, which results in the transmission of impulses to the cerebrum. This somatic sensation and normal function of pain, referred to as nociception or nociceptive pain, informs the organism of impending tissue damage. Somatic and visceral free nerve endings, termed nociceptors, initially process such pain signals.

[0004] Pain is a subjective experience related to perception of inputs to the central nervous system by a specific class of sensory receptors known as nociceptors. Nociceptors fire in response to noxious thermal, mechanical and chemical stimuli. Coding of a stimulus as painful occurs at several levels in the nervous system. The first is at the level of transduction of the noxious stimulus in the peripheral nerve terminals of the nociceptors. During the transduction step, the noxious stimulus is converted to an electrical stimulus in the form of an action potential. In mammals the vanilloid receptors (VR-1 and VRL-1) are proposed to function during transduction of a noxious heat stimulus.

[0005] The second level of coding occurs in the dorsal horn of the spinal cord. The cell bodies of nociceptive neurons are found in the dorsal root ganglia and send projections both to the periphery and to the dorsal horn. Upon stimulation nociceptors release the excitatory neurotransmitter glutamate which produces action potential sin post-synaptic cells of the dorsal horn, which project to the brain where pain is perceived.

[0006] In general, while brain pathways governing the perception of pain are still incompletely understood, sensory afferent synaptic connections to the spinal cord, termed "nociceptive pathways" have been studied. The nociceptive pathway, which exists for protection of the organism (such as the pain experienced in response to a burn), is inactive. Activity is initiated by the application of a high intensity, potentially damaging stimulus.

[0007] Nerve growth factor (NGF) was the first neurotrophin to be identified, and its role in the development and survival of both peripheral and central neurons has been well characterized. NGF has been shown to be a critical survival and maintenance factor in the development of peripheral sympathetic and embryonic sensory neurons and of basal forebrain cholinergic neurons (Smeyne, et al, Nature 368:246-249 (1994); Crowley, et al, Cell 76: 1001-1011 (1994)). NGF upregulates expression of neuropeptides in sensory neurons (Lindsay, et al, Nature 337:362-364 (1989)), and its activity is mediated through two different membrane -bound receptors, the TrkA tyrosine kinase receptor and the p75 receptor which is structurally related to other members of the tumor necrosis factor receptor family (Chao, et al, Science 232:518-521 (1986)).

[0008] In addition to its effects in the nervous system, NGF has been increasingly implicated in processes outside of the nervous system. For example, NGF has been shown to enhance vascular permeability in the rat (Otten, et al., Eur J. Pharmacol. 106: 199-201 (1984)), enhance T- and B-cell immune responses (Otten, et al, Proc. Natl. Acad. Sci. USA 86: 10059-10063 (1989)), induce lymphocyte differentiation and mast cell proliferation and cause the release of soluble biological signals from mast cells (Matsuda, et al, Proc. Natl. Acad. Sci. USA 85:6508-6512 (1988); Pearce, et al. J. Physiol. 372:379-393 (1986);

Bischoff, et al. Blood 79:2662-2669 (1992); Horigome, et al, J. Biol. Chem. 268: 1.4881- 14887 (1993)). Although exogenously added NGF has been shown to be capable of having all of these effects, it is important to note that it has only rarely been shown that endogenous NGF is important in any of these processes in vivo (Torcia, et al, Cell. 85(3):345-56 (1996)).

[0009] NGF is produced by a number of cell types including mast cells (Leon, et al, Proc. Natl. Acad. Sci. USA 91 :3739-3743 (1994)), B-lymphocytes (Torcia, et al, Cell 85:345-356 (1996), keratinocytes (Di Marco, et al, J. Biol. Chem. 268:22838-22846)), smooth muscle cells (Ueyama, et al, J. Hypertens. 11 : 1061-1065 (1993)), fibroblasts (Lindholm, et al, Eur. J. Neurosci. 2:795-801 (1990)), bronchial epithelial cells (Kassel, et al, Clin, Exp. Allergy 31 : 1432-40 (2001)), renal mesangial cells (Steiner, et al, Am. J. Physiol. 261 :F792-798 (1991)) and skeletal muscle myotubes (Schwartz, et al, J Photochem, Photobiol. B 66: 195- 200 (2002)). NGF receptors have been found on a variety of cell types outside of the nervous system. For example, TrkA has been found on human monocytes, T- and B-lymphocytes and mast cells.

[0010] An association between increased NGF levels and a variety of inflammatory conditions has been observed in human patients as well as in several animal models. These include systemic lupus erythematosus (Bracci-Laudiero, et al, Neuroreport 4:563-565 (1993)), multiple sclerosis (Bracci-Laudiero, et al, Neurosci. Lett. 147:9-12 (1992)), psoriasis (Raychaudhuri, et al., Acta Derm, l'enereol. 78:84-86 (1998)), arthritis (Falcimi, et al, Ann. Rheum. Dis. 55:745-748 (1996)), interstitial cystitis (Okragly, et al, J. Urology 161 :438-441 (1991)), asthma (Braun, et al, Eur. J Immunol. 28:3240-3251 (1998)), pancreatitis, and prostatitis.

[0011] NGF mediated signal is necessary for survival of developing neurons and differentiation of developing and mature neurons. NGF is also involved in nociceptive signal processing of afferent neurons in adults. Treatment with exogenous NGF has been shown to lead to an increase in pain and pain sensitivity.

[0012] Recently, a missense point mutation, C661T (leading to the amino acid substitution R100W), was identified in the gene encoding NGFP of a Swedish family with a history of congenital insensitivity to pain perception, which best fits to HSAN (Hereditary sensory and autonomous neuropathy) V. Importantly, HSAN V patients do not display mental retardation or cognitive deficits like other congenital neuropathies, suggesting underlying mechanism of R100W signaling may provide clues to separate pain from trophic function of NGF.

[0013] The use of NGF antagonists, such as an anti-NGF antibody, to treat various types of pain, has been described. See, e.g., U.S. Pub. No. 2004/0131615, U.S. Pub. No.

2004/0253244, U.S. Pub. No. 2004/0237124, WO 04/032870, WO 2005/000194, WO 04/073653, and WO 04/058184. Accordingly, a need exists for novel treatments of neurodegenerative diseases that avoid the pain associated with traditional treatments. [0014] All documents referred to herein, or the indicated portions, are hereby incorporated by reference herein. No document, however, is admitted to be prior art to the claimed subject matter.

SUMMARY OF THE INVENTION

[0015] The present invention is based on the discovery that in vivo administration of a therapeutically effective amount of an NGF mutein such as a human NGF mutein may be used to treat neurodegenerative diseases such as Alzheimer's disease, Down syndrome, diabetic neuropathies, and other diseases associated with NGF deficiencies, without eliciting pain associated with NGF treatment.

[0016] Accordingly, the present invention provides a method of treating a

neurodegenerative disease or disorder in a subject. The method includes administering to a subject in need thereof an effective amount of nerve growth factor (NGF), wherein the NGF consists of the human NGF wild-type amino acid sequence with at least one mutation to the amino acid sequence. In one embodiment, the mutation to the NGF amino acid sequence consists of R100W. In another embodiment, the subject is human. In another embodiment, the subject has one or more of neurodegenerative diseases or disorders selected from the group consisting of Alzheimer's disease, Down syndrome, diabetic neuropathies, and other diseases associated with NGF deficiencies. In yet another embodiment, the treating of the disease or disorder results in amelioration of pain in the subject. In yet another embodiment, the mutated NGF is administered to the subject by direct infusion.

[0017] In another aspect, the invention provides kits comprising the compositions of the invention. In one embodiment, the kit further provides instructions for practicing the methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a pictorial diagram showing NGF-mediated sensitization of nociceptive sensory neurons. Nicol, et al, Molecular Interventions. 2007. 7(1): pp. 26-41.

[0019] Figures 2A-2D are a series of pictorial diagrams showing results from the purification of mature mBtNGF. According to the densitometer, maximal estimated percentage of non-biotinylated NGF is less than 0.99 ± 0.33 (%) of input signal in Figure 2D. [0020] Figures 3A-3C are a series of pictorial and graphical diagrams showing results from analysis of biological activities of mBtNGF using PC 12 cells.

[0021] Figures 4 A and 4B are a series of pictorial diagrams showing results from downstream TrkA signaling stimulated by R100W or mBtNGF.

[0022] Figures 5A-5D are a series of pictorial and graphical diagrams showing results from transport of R100W-QD655 and mBtNGF-QD655 in axon of DRG.

[0023] Figures 6A and 6B are pictorial diagrams showing the structure of the human NGFB protein, adapted from Ryden and Ibenez (J. Biol. Chem., 272, 33085-33091, 1997), with the alternative amino acid in position 100 of the mature protein, highlighted. Figure 6B provides a sequence alignment of amino acids 189-239 of the pro forms of the NGFB proteins from different species (i.e., human (SEQ ID NO: 2), pig (SEQ ID NO: 3), mouse (SEQ ID NO: 4), rat (SEQ ID NO: 5), guinea pig (SEQ ID NO: 6), chicken (SEQ ID NO: 7), and bovine (SEQ ID NO: 8), as well as of human neurotrophins (i.e., NT4/5 (SEQ ID NO: 9), NT3 (SEQ ID NO: 10), and BDNF (SEQ ID NO: 11) showing the conservation of the mutated amino acid in position 100 of the mature NGFB protein (boxed).

[0024] Figure 7A provides a sequence alignment showing the amino acid sequences of the proforms of NGFR100W (SEQ ID NO: 1) and wild-type human NGF (SEQ ID NO: 2). Figure 7B provides the nucleic acid sequence of the pro form of human wild-type NGFB (GenBank X52599.1).

[0025] Figures 8A and 8B are pictorial diagrams showing that NGFR100W binds to 3T3- TrkA cells. To investigate why NGFR100W does not stimulate downstream of p75NTR, internalization assays were performed through TrkA and p75NTR. Because PC 12 has both TrkA and p75NTR receptors, 3T3 was transfected to have either TrkA or p75NTR. Two additional mutants (Δ9/13 and KKE) were cloned in the expression system to

monobiotinylate them; Δ9/13 is known to bind poorly TrkA while maintaining affinity to p75NTR , KKE mutant abolishes p75NTR binding while maintaining affinity to TrkA. Thus, these Δ9/13 and KKE were used as positive controls for p75 and TrkA, respectively. Mono biotinylated NGF is conjugated with streptavidin-Qdots 655 and was applied to starved cells at InM, incubated at 37 for about 30min. wtNGF and KKE, NGFR100W was internalized through TrkA in receptor specific manner as shown in cold competition. KKE =

K32A/K34A/E35A. Δ9/13 = deletion of sequence from 9 to 13. [0026] Figures 9A and 9B are pictorial diagrams showing that NGFR100W does not bind to 3T3-p75NTR cells (shown at 1 nM, but 2 hours). NGFR100W failed to be internalized through p75NTR, while wtNGF and Δ9/13 showed bright fluorescence blinking inside 3T3-p75NTR showing typical pattern of internalization through p75. These findings indicate NGFR100W fails to be internalized through p75NTR leading to failure of stimulating p75NTR downstream.

[0027] Figure 10 is a pictorial diagram showing that NGFR100W fails to hyper-sensitize DRG neurons. Triturated El 5- 16 DRGs were cultured on poly-lys coated cover slips. The media was changed to recording media after 2hrs of starvation. The recordings were only done for small to medium diameter DRGs. Voltage clamp analysis was performed at -60 mV holding potential (V=IR). Low pH (5.5) solution was puffed onto the cell body to evoke current. Following the application of low pH solution, either wt or R100W was applied at 50ng/ml for 10 mins. The identical pulse of low pH was applied and currents were measured. Picospritzer; 30-50ms.

[0028] Figures 11 A and 1 IB are graphical diagrams showing that NGFR100W does not hyper-sensitizes DRG neurons. Wt: 10 min of wtNGF produce acute sensitization shown by and almost three to five fold increase in current. R100W: NGFR100W failed to induce a sensitized response and even showed decreased currents after application. Collectively, the data suggest that NGFR100W lose pain perception by failing potentiation of nociceptive channel through a mechanism involving p75 NTR downstream.

[0029] Figure 12 is a graphical diagram showing NGF-p75 -Ceramide in Pain.

DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention is based on the discovery that in vivo administration of a therapeutically effective amount of an NGF mutein such as a human NGF mutein may be used to treat neurodegenerative diseases such as Alzheimer's disease, Down syndrome, diabetic neuropathies, and other diseases associated with NGF deficiencies, without eliciting pain associated with NGF treatment. The invention is based on the observation that an NGF mutein that carries a point mutation (R100W in the mature NGFB sequence) retains its trophic support activity but no longer elicits a nociceptive response {i.e., pain).

[0031] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[0032] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "the method" includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0033] The term "comprising", which is used interchangeably with "including,"

"containing," or "characterized by," is inclusive or open-ended language and does not exclude additional, unrecited elements or method steps. The phrase "consisting of excludes any element, step, or ingredient not specified in the claim. The phrase "consisting essentially of limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. The present disclosure contemplates embodiments of the invention compositions and methods corresponding to the scope of each of these phrases. Thus, a composition or method comprising recited elements or steps contemplates particular embodiments in which the composition or method consists essentially of or consists of those elements or steps.

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described.

[0035] As used herein, the term "nerve growth factor" and "NGF" refers to nerve growth factor and variants thereof that retain at least part of the activity of NGF. As used herein, NGF includes all mammalian species of native sequence NGF, including human, canine, feline, equine, or bovine.

[0036] There are two pathways that NGF acts on neurons, namely the TrkA- mediated and the p75 -mediated pathways. The TrkA-mediated pathway is critical for neuronal survival and maintenance while the p75-mediated pathway regulates neuronal death. Both pathways contribute to nociceptive response (pain). Normal wild-type NGF activates both pathways thus causing a pain effect in a subject. While being still capable of acting through the TrkA pathway to support neurons, the inventors observed that the mutein NGFrlOOw no longer engages the p75-mediated pathway. By doing so, the p75-mediated pain reaction no longer exists when the NGFR100W mutein is used.

[0037] "NGF receptor" refers to a polypeptide that is bound by or activated by NGF. NGF receptors include the TrkA receptor and the p75 receptor of any mammalian species, including, but not limited to, human, canine, feline, equine, primate, or bovine.

[0038] A "TrkA immunoadhesin" refers to a soluble chimeric molecule comprising a fragment of a TrkA receptor, for example, the extracellular domain of a TrkA receptor and an immunoglobulin sequence, which retains the binding specificity of the TrkA receptor.

[0039] "Biological activity" of NGF generally refers to the ability to bind NGF receptors and/or activate NGF receptor signaling pathways. Without limitation, a biological activity includes any one or more of the following: the ability to bind an NGF receptor (such as p75 and/or TrkA); the ability to promote TrkA receptor dimerization and/or autophosphorylation; the ability to activate an NGF receptor signaling pathway; the ability to promote cell differentiation, proliferation, survival, growth, migration and other changes in cell physiology, including (in the case of neurons, including peripheral and central neuron) change in neuronal morphology, synaptogenesis, synaptic function, neurotransmitter and/or neuropeptide release and regeneration following damage; and the ability to treat or prevent neurodegenerative diseases and disorders.

[0040] As used herein, "treatment" is an approach for obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: improvement in any aspect of pain including lessening severity, alleviation of one or more symptoms associated with a neurodegenerative disease or disorder (e.g., pain associated Alzheimer's disease, Down syndrome, diabetic neuropathies, and other diseases associated with NGF deficiencies) and/or treatment of such a disease or disorder.

[0041] An "effective amount" is an amount sufficient to effect beneficial or desired clinical results including alleviation or reduction in pain. For purposes of this invention, an effective amount of an NGF mutein is an amount sufficient to treat, ameliorate, reduce the intensity of or prevent pain associated with a neurodegenerative disease or disorder and/or a treatment of such a disease or disorder. In some embodiments, the "effective amount" may reduce the pain of ongoing pain and/or breakthrough pain (including ambulatory pain and touch-evoked pain), and it may be administered before, during, and/or after any additional treatments for the neurodegenerative disease or disorder. In another embodiment, the "effective amount" is an amount sufficient to delay development of pain associated with a neurodegenerative disease or disorder and/or any treatment of such a disease or disorder.

[0042] "Reducing incidence" of pain means any of reducing severity (which can include reducing need for and/or amount of (e.g., exposure to) other drugs and/or therapies generally used for these conditions), duration, and/or frequency (including, for example, delaying or increasing time to pain associated with a neurodegenerative disease or disorder and/or a treatment of such disease or disorder.

[0043] As used herein, "co-administration" includes simultaneous administration and/or administration at different times. Co-administration also encompasses administration as a co- formulation (i.e., the NGF antagonist and an agent are present in the same composition) or administration as separate compositions. As used herein, co -administration is meant to encompass any circumstance wherein an agent and NGF mutein are administered to an individual, which can occur simultaneously and/or separately. As further discussed herein, it is understood that the NGF mutein and an agent can be administered at different dosing frequencies or intervals. For example, an NGF mutein of the invention can be administered weekly, while the agent can be administered more frequently. It is understood that the NGF mutein and the agent can be administered using the same route of administration or different routes of administration.

[0044] In humans and other vertebrates, painful stimuli are sensed by specialized neurons known as nociceptors, which fire in response to noxious temperature and mechanical or chemical stimuli, all of which have the potential to cause tissue damage. The signals are in turn processed by the central nervous system and perceived as pain, serving an indispensable protective role. Nociceptors are also involved in pathological pain states caused by inflammation, nerve damage, or cancer. An increased understanding of nociception therefore is of wide interest, and model systems for molecular genetic analysis are desirable.

[0045] Accordingly, in one aspect, the invention provides a method of treating a neurodegenerative disease or disorder. The method includes administering to a subject in need such treatment an effective amount of nerve growth factor (NGF) mutein, wherein the NGF mutein consists of the human wild-type amino acid sequence with at least one mutation to the amino acid sequence. In one embodiment, the mutation is R100W. As such, the invention likewise provides use of the NGF mutein of the invention for treating a

neurodegenerative disease or disorder.

[0046] As used herein, a "mutein" refers to any protein arising as a result of a mutation. Included in the term "mutein" are proteins with altered amino acid sequences as compared to the wild-type protein from which the mutated protein is derived. Without being bound by theory, the amino acid sequence of a mutein is typically sufficiently altered so as to result in a protein having altered properties as compared to the wild-type protein.

[0047] The term "subject" as used herein refers to any individual or patient to which the subject methods are performed. Generally the subject is human, although as will be appreciated by those in the art, the subject may be an animal. Thus other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject.

[0048] The term "pharmaceutically acceptable," when used in reference to a carrier, is meant that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0049] The terms "administration" or "administering" are defined to include an act of providing a compound or pharmaceutical composition of the invention to a subject in need of treatment. The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the subject's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration or administration via intranasal delivery. In one embodiment, the mutein of the invention is administered by direct infusion.

[0050] The term "agonist" refers to an agent or analog that binds productively to a receptor and mimics its biological activity. The term "antagonist" refers to an agent that binds to receptors but does not provoke the normal biological response. Agonists or antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or any other molecules which decrease the normal biological response. An exemplary antagonist of NGF includes, but is not limited to, the human wild-type NGF amino acid sequence having the point mutation of R100W (i.e., an NGF mutein of the invention).

[0051] As used herein, "wild-type NGF" or "wtNGF" and equivalents thereof are used interchangeably and mean native or recombinant human nerve growth factor. It should be understood that NGF is made as a precursor (i.e., "pro form") within a cell, and upon secretion from the cell, the pro sequence is cleaved, resulting in the "mature" form (i.e., amino acids 122-290) of the sequence. Accordingly, while Figure 7 shows a sequence alignment of the proforms of wtNGF (SEQ ID NO: 2) and NGFR100W(SEQ ID NO: 1), showing an amino acid substitution of tryptophan (Trp, W) at position 221 for naturally occurring arginine (Arg, R). However, upon cleavage of the pro sequences, the point mutation of the mature sequence (i.e., amino acids 122-290 of SEQ ID NO: 1) is referred to as R100W (i.e., a substitution of the arginine at position 100 for tryptophan.

[0052] Accordingly, in one aspect, the invention provides a nerve growth factor (NGF) mutein numbered in accordance with human wild-type NGF, wherein the mutein comprises at least one amino acid substitution at position 100 of the mature form thereof. In one embodiment, the substitution is R100W. Thus, the invention provides an isolated nerve growth factor (NGF) mutein consisting of amino acids 122-290 of SEQ ID NO: 1.

[0053] The invention also provides polynucleotides encoding modified NGF muteins. These polynucleotides can be used, for example, to produce quantities of the NGF muteins therapeutic use. The invention further provides degenerative DNA sequences that encode the same NGF muteins. Methods of constructing and expressing degenerative DNA sequences capable of expressing the same amino acid sequence as a given polynucleotide sequence are known in the art. A polynucleotide of the invention can be readily obtained in a variety of ways including, without limitation, chemical synthesis, cDNA or genomic library screening, expression library screening, and/or PCR amplification of cDNA.

[0054] In another aspect, the present invention provides a method of ameliorating or treating pain in a subject by administering the mutein of the invention to the subject. As used herein, the term "ameliorating" or "treating" means that the clinical signs and/or the symptoms associated with anorexia are lessened as a result of the actions performed. The signs or symptoms to be monitored will be characteristic of the particular neurodegenerative disease or disorder being treated, and will be well known to the skilled clinician, as will the methods for monitoring such signs and conditions. Thus, in a similar aspect, the invention provides use of a NGF mutein of the invention for treating or ameliorating pain in a subject.

[0055] As used herein, the terms "reduce" and "inhibit" are used together because it is recognized that, in some cases, a decrease can be reduced below the level of detection of a particular assay. As such, it may not always be clear whether the expression level or activity is "reduced" below a level of detection of an assay, or is completely "inhibited."

Nevertheless, it will be clearly determinable, following a treatment according to the present methods, that the level of pain is at least reduced from the level prior to treatment.

[0056] In another aspect, the invention provides a method for producing mono- biotinylated NGF (mBtNGF). The method includes inserting Avi tag (biotin acceptor peptide, 15 amino acid sequence recognized by E.coli biotin ligase, BirA) into the C-terminal of full length human preproNGF cDNA in pcDNA 3.1 mammalian expression vector. BirA is also subcloned into pcDNA3.1 and transiently co-expressed in HEK293FT with NGF-avi. The prepro sequence of NGF was subsequently processed within the cells and only the mature form is secreted into media, which was purified using Ni-resin. The inventors observed that 1) the NGF produced retained biological activities that were comparable to normal NGF purified from mouse sub maxillary glands; 2) the NGF is biotinylated with only one biotin at the C-terminus by design and the efficiency of biotinylation is high, with virtually undetectable levels of non-biotinylated NGF; and 3) streptavidin-quantum dots were conjugated with biotinylated NGF for single molecule tracking of axonal transport using microfluidic chambers.

[0057] The functional properties of the mature form of NGF R100W mutein (NGFR100W) were then compared with the mature form of wild type NGF (wtNGF). The axonal trafficking was compared using microfluidic culture of dorsal root ganglions (DRG) and TrkA downstream signaling. To determine the binding and internalization through TrkA or p75NTR, KKE (abolishes p75NTR binding) and delta 9/13 (induces 100-fold reduction in TrkA binding) NGF mutants were also cloned using the same method to produce mono- biotinylated mature NGF mutants. The goal was to determine whether NGFR100W fails to elicit p75NTR-mediated signaling linked to pain, while supporting trophic function through TrkA-mediated signaling.

[0058] In another aspect, the methods of the invention are useful for providing a means for practicing personalized medicine, wherein treatment is tailored to a subject based on the particular characteristics of the neurodegenerative disease or disorder of the subject. The method can be practiced, for example, by administering a mutein of the invention to a subject having signs or symptoms of a neurodegenerative disease or disorder, wherein a decrease in pain associated with the disease or disorder, or treatment of the disease or disorder, following administration of the mutein identifies the mutein as useful for treating the disease or disorder. Likewise, the mutein may be administered in combination with an additional agent used for treating the neurodegenerative disease or disorder and/or for treating pain in general. Any decrease in pain associated with the disease or disorder, or treatment thereof, following administration of the combination identifies the agent as useful for treating the disease or disorder.

[0059] Once disease is established and a treatment protocol is initiated, the methods of the invention may be repeated on a regular basis to evaluate whether the level of pain in the subject begins to approximate that which is observed in a normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months. Accordingly, the invention is also directed to methods for monitoring a therapeutic regimen for treating a subject having a neurodegenerative disease or disorder. A comparison of the level of pain and/or the signs and symptoms associated with the neurodegenerative disease or disorder prior to and during therapy indicates the efficacy of the therapy. Therefore, one skilled in the art will be able to recognize and adjust the therapeutic approach as needed.

[0060] All methods may further include the step of bringing the active ingredient(s) into association with a pharmaceutically acceptable carrier, which constitutes one or more accessory ingredients. Pharmaceutically acceptable carriers useful for formulating an agent for administration to a subject are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters. A pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize or to increase the absorption of the conjugate. Such physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the physico-chemical characteristics of the therapeutic agent and on the route of administration of the composition, which can be, for example, orally, intranasally or any other such method known in the art. The pharmaceutical composition also can contain a second (or more) compound(s) such as a diagnostic reagent, nutritional substance, toxin, or therapeutic agent, for example, a cancer chemotherapeutic agent and/or vitamin(s). Thus, an aspect of the invention provides use of the NGF muteins of the invention in a medicament for treating or ameliorating pain in a subject.

[0061] The total amount of a compound or composition to be administered in practicing a method of the invention can be administered to a subject as a single dose, either as a bolus or by infusion over a relatively short period of time, or can be administered using a fractionated treatment protocol, in which multiple doses are administered over a prolonged period of time. One skilled in the art would know that the amount of the mutein of the invention to treat a neurodegenerative disease or disorder in a subject depends on many factors including the age and general health of the subject as well as the route of administration and the number of treatments to be administered. In view of these factors, the skilled artisan would adjust the particular dose as necessary. In general, the formulation of the pharmaceutical composition and the routes and frequency of administration are determined, initially, using Phase I and Phase II clinical trials.

[0062] In another aspect, the invention provides a composition for treating a

neurodegenerative disease or disorder. The composition includes an effective amount of a nerve growth factor (NGF) mutein, in combination with one or more pharmaceutically acceptable excipients. In one embodiment, the NGF mutein consists of the mature human wild-type NGF sequence with the point mutation R100W. In another embodiment, the NGF mutein is co-administered with an agent for treating pain (e.g., an opioid analgesic). In yet another embodiment, the NGF mutein is co-administered with an agent for treating the neurodegenerative disease or disorder. Thus, the invention also provides use of the NGF muteins of the invention in a medicament for treating a neurodegenerative disease in a subject.

[0063] In another aspect, the invention provides kits for performing the methods of the invention that include an NGF mutein of the invention. In one embodiment, the invention provides a kit that includes a pharmaceutical composition comprising a mutein of human NGF, wherein the wild-type amino acid sequence contains a point mutation consisting of R100W. In another embodiment, the kit includes instructions for practicing the methods of the invention.

[0064] The following examples are intended to illustrate but not limit the invention.

EXAMPLE 1

NGFRIOOW BINDS T0 TRKA

[0065] To investigate why NGFR100W does not stimulate downstream of p75NTR, an internalization assay was performed either through TrkA or p75NTR. To distinguish between TrkA and p75NTR receptors, NIH 3T3 cells that express either TrkA or p75NTR were utilized in binding and internalization assays. The wild-type NGF (wtNGF) was used as a positive control for both TrkA and p75NTR. Additionally, two known NGF mutants were subcloned in the expression system and monobiotinylated proteins were purified: the Δ9/13 mutant (Δ9/13 = deletion of sequence from 9 to 13) is known to bind poorly to TrkA, while maintaining an affinity to p75NTR (Hughes et al., 2001); the KKE mutant (KKE = K32A/K34A/E35A ) abolishes p75NTR binding, while maintaining an affinity to TrkA (Mahapatra et al., 2009). Thus, KKE and Δ 9/13 were used as positive controls for TrkA and p75NTR, respectively.

[0066] To visualize binding and internalization of NGF, a method was developed to produce biotinylated NGF that contains single biotin at a specific site, which was proven not to hinder its biological function (Sung et al. 201 1). Monobiotinylated NGF (wt, R100W, Δ9/13 and KKE) was conjugated with streptavidin-Qdots 655 (QD655). NIH 3T3-TrkA or - p75NTR cells were serum- starved and were incubated with 1 nM mBtNGF-QD655 (wt, R100W, Δ9/13 and KKE) at 37°C for 30 min. Cells were extensively washed and live imaging was carried out to examine binding and internalization of mBtNGF-QD655. The results show: 1) the wtNGF, NGFR100W, and the KKE mutant were internalized into NIH

3T3-TrkA cells, while the Δ9/13 was not; 2) the result of a cold completion experiment with an excessive amount of non-labeled wtNGF demonstrated that binding and internalization of NGFRioow int0 NIH3T3.TrkA cells was completely inhibited under this condition; and 3) QD655 only yielded undetectable signals, as expected (Figures 8A and 8B).

[0067] Similar experiments were conducted using NIH 3T3-p75NTR cells and the results showed that: 1) the wtNGF and the Δ9/13 mutant were internalized into these cells; 2) NGFR100W and the KKE mutant failed to be internalized; and 3) again, a cold completion experiment and QD655 only gave rise to an un-detectable signal (Figures 9A and 9B).

[0068] Taken together, these results suggest that NGFR100W exhibited behaviors similar to the KKE mutant, but very different from the Δ9/13 mutant. It was therefore concluded that NGFR100W retains its ability to bind and signal through TrkA, but fails to bind and signal through p75NTR.

EXAMPLE 2

NGFRIOOW Does Not Elidt Hyper-Sensitization of Sensory Neurons

[0069] Among known mechanisms of pain transduction, NGF produces acute

hypersensitivity to heat or mechanical stimuli by potentiating nociceptive channel, TRPV1. Indeed, some members of the TRP channel family are activated by PLC-γ coupled

mechanism. Therefore, decreased signal of p75NTR downstream including PLC-γ may reduce NGF mediated potentiation when DRG was stimulated by NGFR100W.

[0070] Single neuron patch clamp assays were performed using E15-16 rat dorsal root ganglion neurons (DRG), which contain endogenous TRP VI (Figure 10). Current was measured after application of either wtNGF or NGFR100W. Because TRPVl is a cation channel that is activated by extracellular protons, a moderate acidic solution (pH 5.5) was puffed around the patch clamped cell body. The resulting proton-evoked current was measured and compared before and after 10 min. application of either NGF or NGFR100W. Previous studies have shown that 10 min. of NGF application to DRGs sensitized a capsaicin- evoked response (Shu and Mendell, 1999). As shown herein, it was similarly observed that 10 min of wtNGF exposure produced acute sensitization shown by an almost two-fold increase in current (Figures 11 A and 1 IB). However, NGFR100W failed to induce a sensitized response and even showed decreased currents after application. The relative change in charge transfer was measured and the results show that the value was -2.90 for wtNGF, 0.2 for NGFR100W (p<0.05) (Figures 11 A and 1 IB). Collectively, the data presented herein suggests that NGFR100W fails to cause hyper-potentiation of nociceptive channel through a yet-undefined mechanism involving p75NTR downstream.

EXAMPLE 3

Ceramide is Identified as a Contributor to Hyper-Sensitization of Sensory Neurons

[0071] To investigate what signaling molecule(s) that may contribute to hyper- sensitization of DRG neurons induced by NGF, DRG neurons were pretreated with 1 μΜ ceramide analog (C2) followed by treatment with NGFR100W, 5 μΜ PLCy inhibitor (U73122) or 10 μΜ neutral sphingomyelin inhibitor (manumycin). As shown above, wtNGF caused hyper-potentiation while NGFR100W failed to do so. Strikingly, pretreatment with C2 ceramide analog significantly restored hyper-potentiation. Both the PLCy inhibitor and the neutral sphingomyelin inhibitor also showed an inhibitory effect, but to a much lesser degree (Figure 12). These data suggest that ceramide production following NGF binding to p75NTR may contribute prominently to hyper-sensitization of sensory neurons.

[0072] References

[0073] Hughes, et al. 2001. Distinction between differentiation, cell cycle, and apoptosis signals in PC 12 cells by the nerve growth factor mutant delta9/13, which is selective for the p75 neurotrophin receptor. JNeurosci Res. 63: 10-9.

[0074] Mahapatra, et al. 2009. Identification of critical residues within the conserved and specificity patches of nerve growth factor leading to survival or differentiation. J Biol Chem. 284:33600-13.

[0075] Sung, et al. 2011. A novel method for producing mono-biotinylated, biologically active neurotrophic factors: An essential reagent for single molecule study of axonal transport. JNeurosci Methods. 200: 121-8

[0076] Shu, et al. 1999. Neurotrophins and hyperalgesia. Proc Natl Acad Sci USA. 96:7693-6.

[0077] While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the disclosed principles and including such departures from the disclosure as come within known or customary practice within the art to which the disclosure pertains and as may be applied to the essential features hereinbefore set forth. Accordingly, the invention is limited only by the following claims.

Claims

What is claimed is:
1. A nerve growth factor (NGF) mutein numbered in accordance with human wild-type NGF, wherein the mutein comprises at least one amino acid substitution at position 100.
2. The NGF mutein of claim 1, wherein the substitution is R100W.
3. An isolated nerve growth factor (NGF) mutein consisting of amino acid residues 122- 290 of SEQ ID NO: 1.
4. A method of treating a neurodegenerative disease or disorder comprising
administering to a subject in need thereof an effective amount of nerve growth factor (NGF), wherein the NGF consists of the human wild-type amino acid sequence with at least one mutation to the amino acid sequence, thereby treating the
neurodegenerative disease or disorder of the subject.
5. The method of claim 4, wherein the mutation to the NGF amino acid sequence
consists of R100W (amino acid residues 122-290 of SEQ ID NO: 1).
6. The method of claim 1, wherein the subject is human.
7. The method of claim 6, wherein the subject has one or more of neurodegenerative diseases or disorders selected from the group consisting of Alzheimer's disease, Down syndrome, diabetic neuropathies, and other diseases associated with NGF deficiencies.
8. The method of claim 7, wherein the treating of the disease or disorder results in
amelioration of pain in the subject.
9. The method of claim 6, wherein the mutated NGF is administered to the subject by direct infusion.
10. A method for treating pain comprising administering to a subject in need thereof an effective amount of nerve growth factor (NGF), wherein the NGF consists of the human wild-type amino acid sequence with at least one mutation to the amino acid sequence, thereby treating the neurodegenerative disease or disorder of the subject.
11. The method of claim 10, wherein the mutation to the NGF amino acid sequence consists of R100W (amino acid residues 122-290 of SEQ ID NO: 1).
12. The method of claim 10, wherein the subject is human.
13. The method of claim 12, wherein the subject has one or more of neurodegenerative diseases or disorders selected from the group consisting of Alzheimer's disease, Down syndrome, diabetic neuropathies, and other diseases associated with NGF deficiencies.
14. The method of claim 13, wherein the treating of the disease or disorder results in amelioration of pain in the subject.
15. The method of claim 12, wherein the mutated NGF is administered to the subject by direct infusion.
16. A composition comprising the NGF mutein of claim 1 and a pharmaceutically
acceptable carrier.
17. A composition comprising the NGF mutein of claim 3 and a pharmaceutically
acceptable carrier.
PCT/US2012/040970 2011-06-06 2012-06-06 Compositions and methods for treating neurodegenerative diseases WO2012170452A2 (en)

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