EP1019081A2 - Complexes d'apolipoproteine e/facteur de croissance et procedes d'utilisation - Google Patents

Complexes d'apolipoproteine e/facteur de croissance et procedes d'utilisation

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Publication number
EP1019081A2
EP1019081A2 EP98950816A EP98950816A EP1019081A2 EP 1019081 A2 EP1019081 A2 EP 1019081A2 EP 98950816 A EP98950816 A EP 98950816A EP 98950816 A EP98950816 A EP 98950816A EP 1019081 A2 EP1019081 A2 EP 1019081A2
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EP
European Patent Office
Prior art keywords
apolipoprotein
complex
composition
apoe
ngf
Prior art date
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EP98950816A
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German (de)
English (en)
Inventor
William D. Matthew
Warren J. Strittmatter
Catherine R. Gutman
Stephanie M. Fullerton
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Duke University
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Duke University
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Publication of EP1019081A2 publication Critical patent/EP1019081A2/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/217—IFN-gamma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to compositions comprising complexes containing apolipoprotein E and nerve growth factor, neurotrophin 4, or ⁇ -interferon and methods of administering the same.
  • Apolipoprotein E protein: apoE; allele: APOE
  • apoE protein: apoE; allele: APOE
  • CSF cerebrospinal fluid
  • apoE mRNA expression of apoE mRNA by astrocytes in the hippocampus increases following entorhinal cortex lesion (Poirier et al. , (1991) Mol. Brain Res. 11, 97). Oligodendrocytes and macrophages increase expression of apoE following optic and sciatic nerve injury, respectively (optic: Stoll et al , (1989) GLIA 2, 170; sciatic: Skene and Shooter, (1983) Proc. Nat. Acad. Sci. USA 80, 4169; Stoll and Mueller, (1986) Neurosci. Lett. 72, 233), and apoE protein accumulates to 5% of total extracellular protein following peripheral nervous system (PNS) injury (Skene and Shooter, (1983) Proc. Nat.
  • PNS peripheral nervous system
  • APOE is a susceptibility gene for familial and late-onset Alzheimer's disease (AD: Strittmatter et al. , (1993) Proc. Nat. Acad. Sci. USA 90, 1977; for review see Strittmatter and Roses, (1995) Proc. Nat. Acad. Sci. USA 92, 4725).
  • AD Strittmatter et al.
  • the gene dose of APOE4 one of the three major alleles of APOE in humans, is correlated with increased risk and decreased average age of onset of AD.
  • apoE2 contains a cysteine at position 158 and apoE4 contains an arginine at position 112.
  • ApoE contains two distinct structural and functional domains, a hydrophobic domain and a hydrophilic receptor binding domain (Weisgraber, (1994) Adv. Prot. Chem. 45, 249).
  • the crystal structure of the hydrophilic domain of apoE is homologous to the family of four-helix bundle growth factors, including ciliary neurotrophic factor, although the sequences of these proteins diverge greatly.
  • CNTF reviewed by Bazan, (1991) Neuron 7, 197; Mott and Campbell, (1995) Curr. Opin. Struc. Biol. 5, 114; apoE crystal structure by Wilson et al. , (1991) Science 252, 1817; CNTF crystal structure by McDonald et al. , (1995) EMBO J. 14, 2689.
  • Nerve growth factor belongs to a family of neurotrophins that includes brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), neurotrophin 4/5 (NT4/5), and neurotrophin 6 (NT6). All of these proteins bind to the low affinity NGF receptor p75 and a specific member of the trk family of tyrosine kinase receptors. For NGF, this is the trkA receptor. Binding of the 26 kDa homodimer of NGF to trkA induces an intracellular signaling cascade that begins with autophosphorylation of trkA.
  • BDNF brain-derived neurotrophic factor
  • NT3 neurotrophin 3
  • NT4/5 neurotrophin 4/5
  • NT6 neurotrophin 6
  • trkA activation of trkA ends with changes in gene expression that effect neuronal survival, outgrowth, excitability, and differentiation. It is well-established that NGF promotes survival and neurite outgrowth of sympathetic and neural-crest derived sensory cells during development (for review, see Snider and Johnson, (1989) Annual Neurology 26, 489).
  • mice that are homozygote mutants for either NGF or trkA have severe loss of cells in the superior cervical ganglion (SCG; > 90%) and in dorsal root ganglia (DRG, 70-80%) (Crowley et al , (1994) Cell 78, 1001 ; Smeyne et al , (1994) Nature 368, 246) and die within 3 weeks of birth.
  • SCG superior cervical ganglion
  • DRG dorsal root ganglia
  • the ability of NGF to promote neurite outgrowth is one of the most dramatic activities of NGF. Indeed, neurite outgrowth from explanted embryonic chick DRGs is the standard bioassay to test for NGF activity. Dissociated cultures of DRGs and SCGs also respond to NGF with increased neuritic growth.
  • PC12 cell line derived from rat adrenal medulla, has provided an excellent system in which to study the neurite outgrowth and survival effects of NGF. If PC 12 cells are cultured in medium supplemented with fetal bovine and horse serum, they proliferate and appear as small, round phase-bright cells. Upon addition of NGF to the culture medium, these cells stop dividing and differentiate into a sympathetic-like neuronal phenotype with multiple neuritic processes and large cell bodies. NGF is also required for survival of PC 12 cells in a serum-free environment. Because of the ease of this paradigm, much of the work elucidating the mechanism of NGF action has used PC 12 cells.
  • Exogenous NGF can promote the survival and regeneration of transected sciatic nerves.
  • Multiple studies have confirmed that silastic tube implants filled with NGF enhance the rate and degree of functional sensory recovery. Rich et al ,
  • Neurotrophin 4 is another member of the neurotrophin family. NT4 has been reported to promote survival of corticospinal motor neurons from neonatal rats (Junger and Varon, (1997) Brain Res. 762, 56), increase survival of cultured rat septal neurons under normal and stress conditions (Nonner et al, (1996)/. Neurosci. 16, 6665), promote survival and morphological and biochemical differentiation of embryonic rat striatal neurons in culture (Ventimiglia et al. (1995), Eur. J. Neurosci. 7, 213), and enhance survival of cultured rat vestibular ganglion neurons and protect these cells against neurotoxic agents (Zheng et al , (1995) J. Neurobiol. 28, 330).
  • Interferons are proteins secreted by eukaryotic cells after virus infections, which in turn protect against virus infections.
  • Three classes of interferons are known at present: they are referred to as IFN ⁇ , IFN ⁇ and IFN ⁇ , which differ in both their structure and biological effects. All of the IFN ⁇ identified thus far are glycosylated, although glycosylation does not appear to influence biological activity.
  • Keller et al (1983) J. Biol. Chem. 258, 8010.
  • Gamma- interferon is regarded as a lymphokine since it is produced by lymphoctyes after either specific or non-specific stimulation by antigens.
  • Gamma-interferon is well- known as an anti- viral and anti-tumor agent. See U.S.
  • Other investigators have found that IFN ⁇ increases intravascular CI inhibitor concentrations in patients exhibiting or at risk for CI inhibitor deficiencies.
  • U.S. Patent No. 5,271,931 to Lotz et al CI inhibitor is a serine protease inhibitor that is involved in the regulation of several proteolytic systems including the complement, contact, coagulation, and fibrinolytic systems. Davis et al. (1988) Ann. Rev. Immunol. 6, 595.
  • Other activities of IFN ⁇ include promotion of polynuclear giant cell formation and activation of macrophages (Weinberg et al. , (1984) Proc. Natl. Acad. Sci.
  • NGF, NT4 and IFN ⁇ possess biologically significant activities.
  • NGF and NT4 neurotrophic growth factors
  • apoE binds to and potentiates the biological action of the neurotrophic factor NGF.
  • potentiate it is meant that apoE enhances at least one biological action of NGF, by increasing the maximal response achieved and/or increasing the potency of the growth factor (i.e. , shifting the dose-response curve to the left).
  • the term “potentiate” indicates that apoE stabilizes the NGF molecule and decreases its degradation.
  • apoE advantageously binds to NT4 and IFN ⁇ .
  • the present invention provides a composition comprising a complex of apoE and NGF.
  • compositions comprising a complex of apoE and NT4.
  • compositions comprising a complex of apoE and IFN ⁇ .
  • the apoE component can be apoE2, apoE3, or apoE4, and can be in the native lip id-bound or a delipidated state.
  • the complex can be formed by either covalent or noncovalent interactions between the apoE and NGF, NT4 or IFN ⁇ molecules.
  • the present invention provides a method of enhancing the survival of neural cells, comprising administering to the neural cells a survival- enhancing amount of a composition comprising a complex of apoE and NGF or NT4.
  • the present invention provides a method of administering a composition comprising a complex of apoE and NGF or NT4 in a therapeutically- effective amount to a subject in need thereof.
  • the present invention provides a method of protecting cells against virus infection, comprising administering to the cells an anti-virus effective amount of a composition comprising a complex of apoE and IFN ⁇ .
  • the present invention provides a method of administering IFN ⁇ in an anti-tumor effective amount to a subject in need thereof.
  • Figure 1 demonstrates that apoE3 potentiates the survival promoting activity of NGF in PC 12 cells.
  • the data are presented as the mean percent survival ⁇ standard deviation as compared to the control treatment of 1000 ng/ml NGF and no apoE.
  • Student's t test for the difference between the means, p ⁇ 0.001 at 100 and 1000 ng/ml NGF.
  • Open bars substrate-bound apoE3 (50 ng/ml) preincubated with 0, 100 and 1000 ng/ml NGF.
  • Black bars soluble NGF alone added at 0, 100 and 1000 ng/ml.
  • Figure 2 demonstrates that apoE3, but not apoE4, enhances neurite outgrowth from PC12 cells in response to NGF.
  • Neurite outgrowth was measured after 3 days in culture with serum-free medium containing 100 ng/ml NGF and 1 ⁇ g/ml apoE3, apoE4 or bovine serum albumin (BSA) as a control protein.
  • Phase images of cells were digitally collected on random fields using IMAGE 1" analysis software (Universal Imaging Corp.; West Chester, PA). Neurite length is displayed as percent of outgrowth of the control treatment (NGF + BSA) ⁇ SEM.
  • NGF + BSA control treatment
  • Figure 3 demonstrates that apoE knockout (KO) mice have a reduced number of unmyelinated fibers as compared with wildtype (WT) mice.
  • the total area covered by high-powered electron micrographs from 3 wildtype and 3 knockout animals was approximately 18,000 ⁇ m 2 for each group (17,900 for wildtype and 18,200 for knockout).
  • the total number of myelinated and unmyelinated axons in this area were counted. It was found that knockout nerves had 50% fewer unmyelinated fibers, but an equal number of myelinated fibers, as compared with nerves from wildtype animals.
  • Figure 4 demonstrates that apoE knockout mice have a delayed response to noxious thermal stimuli.
  • the withdrawal latency in seconds for mice to remove their hind feet from a 55°C water bath was measured.
  • the data presented are the means ⁇ SEM for 3 mice in each group and 3 measurements/per hind foot for each mouse.
  • Knockout mice have over a 50% increase in their response latency as compared with wildtype mice.
  • the p value for the difference between the two means is p ⁇ 0.001 using a paired Student's t test.
  • the nervous system responds to acute injury and to chronic neurodegenerative diseases by the coordinated expression of many proteins, including growth and survival factors, cell surface receptors, and secreted extracellular proteins. Many of these proteins, most notably the neurotrophic factors, play a role in recovery from injury and protection from neurodegenerative disease. Within the complex milieu of the traumatized nervous system it is extremely unlikely that any one growth factor functions alone. Cooperative interactions of growth factors with other proteins are likely to play important roles in the stability, localization or presentation of growth factors. Optimal functioning of growth and survival factors released at sites of injury is critical for recovery from neural trauma. In both the peripheral and central nervous systems the expression of apoE is increased in acute and chronic stress.
  • apoE binds to the neurotrophic factors NGF and NT4. Further disclosed is the discovery that apoE potentiates the biological activity of NGF. As described above, by “potentiate” it is meant that apoE enhances at least one biological action of NGF, by increasing the maximal response achieved and/or increasing the potency of the growth factor (i.e. , shifting the dose-response curve to the left). Alternately, the term “potentiate” indicates that apoE stabilizes the NGF molecule and decreases its degradation.
  • apoE may act as an accessory protein for these growth factors, regulating their metabolism or biological activities.
  • One mechanism may entail the localization of the growth factors to extracellular matrix by tethering them through an apoE molecule. It has previously been shown that apoE binds to the extracellular matrix protein laminin, and thereby increases neuron adhesion, and alters growth cone spreading (Huang et al, (1995) Exp. Neurology 136, 251). ApoE may additionally alter the biological activity of the bound growth factor by other mechanisms, such as inhibiting its proteolytic inactivation, or by altering its ability to interact with its cell-surface receptor. Interactions between apoE and NGF and/or NT4 may be important in modulating the role of these growth factors in neural regeneration responses, both in acute and in chronic disorders of the nervous system.
  • compositions containing apoE:NGF and apoE:NT4 complexes can be formed by simply mixing apoE and the neurotrophic factor(s) (typically aqueous solutions) together, or by any other suitable method known in the art.
  • the compositions can contain both complexes of apoE with NGF and complexes of apoE with NT4.
  • compositions containing the claimed apoE complexes produced in this manner will generally contain apoE, NGF and/or NT4 monomers, homodimers and multimers. In general, as low as 35% , 25 % , 20% , 15% , or lower, or as high as 40% , 50% , 70% , 85 % , 90% , 95% , 99% or higher of the NGF or NT4 molecules in the composition will be complexed with apoE.
  • apoE monomers, homodimers, homotrimers, etc. may associate with NGF or NT4 monomers, homodimers, homotrimers, etc.
  • the present invention encompasses complexes between one or more apoE molecules with one or more NGF or NT4 molecules.
  • the active form of NGF is a homodimer, and it is believed that the NGF homodimer complexes with an apoE monomer. Heterodimeric apoE:NGF and heterotrimeric apoE:(NGF) 2 complexes are preferred. Likewise, heterodimers of apoE and NT4 are preferred. Complexes between one or more apoE molecules with one or more NT4 molecules are also aspects of the present invention. Those skilled in the art will appreciate that apoE:NGF and apoE:NT4 complexes may also be more loosely associated with additional molecules.
  • apoE may first be bound to a substrate, such as a polymeric surface (i.e. , tissue culture plate, test tube), prior to being exposed to and forming a complex with NGF or NT4.
  • a substrate such as a polymeric surface (i.e. , tissue culture plate, test tube)
  • apoE-bound substrates are useful for collecting NGF or NT4 from a solution.
  • the apoE:NGF or apoE:NT4 complex can be bound to a substrate after complex formation. Such substrates are useful for culturing cells in vitro.
  • the strength of the binding interaction between apoE and NGF or NT4 is high; with a dissociation constant of the apoE complexes of at least 10 "7 , preferably at least 10 "8 , more preferably at least 10 "9 .
  • Complexes can be formed by covalent or noncovalent interactions, with covalent complexes of apoE and NGF or NT4 being preferred.
  • apoE:NGF and apoE:NT4 complexes are stable in a solution containing at least 1 % SDS.
  • the apoE component of the claimed complexes can be apoE2, apoE3, apoE4 or a combination thereof.
  • the complexes contain apoE3.
  • ApoE variants and fragments that bind to the apoE receptor and/or complex with and potentiate the biological actions of NGF and/or NT4 are also encompassed by the present invention.
  • ApoE can be from any species of origin, preferably of mammalian origin, more preferably human origin.
  • the apoE molecules can be in the native lipid-bound state or a delipidated state, with delipidated being preferred.
  • ApoE can be purified from natural sources (i.e. , blood, serum or peritoneal fluid).
  • ApoE may also be separated from contaminating proteins using heparin- sepharose chromatography, which utilizes the heparin-binding property of apoE. Rail et al , (1986) E. Methods Enzymol 128, 273. ApoE may be isolated and/or purified, optionally to homogeneity, by conventional techniques such as affinity chromatography, size-exclusion chromatography, gas chromatography, HPLC, and combinations thereof. Separation of the non-cysteine containing E4 isoform of apoE from contaminating cysteine-containing proteins may be accomplished using thiopropyl chromatography on thiopropyl Sepharose (Weisgraber et al. (1983), J. Biol Chem.
  • Recombinant apoE can be produced using methods known in the art, and human recombinant apoE is commercially available. However, recombinant protein is not in the native glycosylated form and is subject to denaturation and oxidation during purification.
  • the NGF component of the complex can also be from a native or recombinant source, and can be produced by any means known in the art. Native: U.S. Patent Nos. 5,210,185 and 5,057,223 to Delia Valle et al ; U.S. Patent No. 4,407,744 to Young. Recombinant: U.S. Patent No. 5,272,063 to Chan et al ; U.S. Patent No.
  • NGF component of the claimed apoE:NGF complexes can be from any species of origin, preferably of mammalian origin, more preferably of human origin.
  • the term "NGF” encompasses NGF variants, analogs and derivatives.
  • U.S. Patent No. 5,349,055 to Persson et al. discloses NGF analogs with significantly reduced binding to the low affinity p75 receptor and essentially no alteration in binding to the trk receptor. Further disclosed by this reference are NGF analogs having increased stability.
  • NGF brain derived growth factor
  • the NT4 component of the disclosed apoE:NT4 complexes can be from a native or recombinant source, and can be produced by any means known in the art. Recombinant: U.S. Patent No. 5,364,769 to Rosenthal. Moreover, the NT4 component of the claimed apoE:NT4 complexes can be from any species of origin, preferably of mammalian origin, more preferably of human origin. The term "NT4" also encompasses NGF variants, analogs and derivatives, such as those disclosed in U.S. Patent No. 5,364,769 to Rosenthal and U.S. Patent No. 5,349,055 to Persson.
  • NT4 also includes NT4 fragments that bind to NGF receptors and/or elicit the neurotrophic actions of NT4.
  • Methods of enhancing the survival of neural cells by administration of an apoE:NGF and/or apoE:NT4 complex are also an aspect of the present invention.
  • the term "enhancing the survival” of neural cells is intended to be construed broadly, and includes neurotrophic and neuro-regenerative actions of the claimed compositions.
  • the expression “enhancing the survival” of neural cells refers to the action of the claimed compositions in protecting neural cells from injury and/or improving recovery from neural injury. By “enhancing the survival” it is meant that the inventive complexes provide some improvement to neural cell survival, as defined above.
  • neural cell survival can be 5 % , 10% , 25% , 50% , 75% , 100% or more.
  • Neuronal cells includes the cells and tissues of the central nervous system and the peripheral nervous system, both in vitro and in vivo.
  • Compositions comprising apoE:NGF and/or apoE:NT4 complexes for use in the claimed methods are as described in more detail hereinabove.
  • the claimed apoE complexes can be administered to neural cells in vitro.
  • in vitro administration will simply require adding a solution (i.e. , aqueous) containing the apoE:NGF and/or apoE:NT4 complexes to the culture medium.
  • a solution i.e. , aqueous
  • the apoE and NGF or NT4 components can be added individually, either concurrently or sequentially, to the culture medium.
  • the apoE molecule or the apoE: growth factor complex can be bound to a substrate (i.e. , a tissue culture dish or petri dish), preferably prior to culturing cells on the substrate. It is well-known in the art that apoE binds to polymeric or coated (i.e. , with extracellular matrix proteins such as laminin) surfaces of tissue culture plates.
  • apoE and NGF or NT4 which potentiate the activity of these neurotrophic factors, are useful in vitro for culturing neural cells, such as immortalized PC12 cells (see, e.g. , U.S. Patent No. 5,349,055 to Persson et al.) and primary cultures of neurons (see, e.g. , Varon, (1997) Brain Res. 762, 56; Barrett and Barrett, (1996) J. Neurosci. 16, 6665; Abiru et al , (1996) Brain Res. Dev. Brain Res. 91, 260; Snider and Johnson, (1989) Annual Neurology 26, 489; Yip et al , (1984) J. Neurosci. 4, 2986).
  • neural cells such as immortalized PC12 cells (see, e.g. , U.S. Patent No. 5,349,055 to Persson et al.) and primary cultures of neurons (see, e.g. , Varon, (1997) Brain Res
  • NGF additionally finds use as a component of culture medium to enhance the survival of neurons. Also disclosed is the use of NGF to provide a therapeutic treatment for Alzheimer's disease, Huntington's disease, and other neurodegenerative disorders. As established by U.S. Patent No. 5,604,202 to Kessler et al , NGF also finds use in methods of treating drug-induced neuropathy. Thus, the presently claimed NGF complexes possessing enhanced neurotrophic activity find similar uses as previously known NGF compositions.
  • NT4 finds use as a component of culture media to enhance the survival or induce the outgrowth of nerve cells in vitro. Also disclosed is use of NT4 in the therapy of neurodegenerative diseases. Thus, the presently claimed NT4 complexes possessing enhanced neurotrophic activity find similar uses as previously known NT4 compositions. The claimed apoE:NGF and apoE:NT4 complexes also find use in tissue culture, because the complexed growth factors are protected against proteolytic degradation.
  • the claimed complexes find use in methods of quantifying or purifying NGF or NT4 receptors or other proteins that bind to these neurotrophic factors.
  • ApoE is readily bound to surfaces, such as test tubes and microtiter plates.
  • NGF or NT4 can be tethered to surfaces for use in sandwich assays or affinity purification techniques, which are well-known to those skilled in the art.
  • the claimed apoE:NGF or apoE:NT4 complexes can also be administered to a subject in vivo. Methods of administration and pharmaceutical formulations of the claimed compositions are described in more detail hereinbelow.
  • the memods of the invention are useful for treating a subject afflicted with a neurodegenerative disease and subjects who have experienced injury or trauma to neural tissue. Such subjects include but are not limited to those afflicted with Alzheimer's Disease, Parkinson's Disease, peripheral nerve injury, peripheral neuropathy (in particular, diabetes- induced peripheral neuropathy), amyotrophic lateral sclerosis, head injury, and stroke.
  • the present invention is particularly useful in treating Alzheimer's Disease and diabetes-induced peripheral neuropathy.
  • Subjects suitable for carrying out the present invention are, in general, mammals and avians, including but not limited to humans, monkeys, equines, caprines, bovines, ovines, porcines, dogs, cats, rabbits, rats, hamsters, mice, quail, chickens and turkeys. Human subjects are presently preferred for in vivo administration.
  • cultured neural tissues/cells for use with the inventive methods include mammalian and avian tissues and cells, including but not limited to neural tissues and cells derived from humans, monkeys, equines, caprines, bovines, ovines, porcines, dogs, cats, rabbits, rats, hamsters, mice, quail, chickens and turkeys.
  • compositions may be contained in a physiologically acceptable carrier (preferably sterile), which is a carrier that is not unduly detrimental to cells and includes pharmaceutically acceptable carriers.
  • a physiologically acceptable carrier preferably sterile
  • the inventive compositions are typically admixed with a pharmaceutically acceptable carrier.
  • the carrier will typically be a liquid.
  • the carrier may be either solid or liquid, such as sterile, pyrogen-free water or sterile pyrogen-free phosphate-buffered saline solution.
  • one may incorporate or encapsulate the claimed complexes in a suitable polymer matrix, liposome or membrane, thus providing a sustained-release delivery device suitable for implantation near the site to be treated locally.
  • compositions for administration to a subject use can be made of pharmaceutical carriers adapted for conventional forms of administration, for example, injection solutions, tablets, capsules, dragees, syrups, solutions, suspension and the like.
  • an injection medium it is preferred to use water which contains the additives usual in the case of injection solutions, such as stabilizing agents, salts or saline, and/ or buffers.
  • the active agent or its pharmaceutical formulation may be contained within a nutritional medium, e.g. , in nutritional supplements.
  • Oral formulations may be slow release preparations or enteric coated preparations to facilitate delivery of the peptide to the small intestine.
  • any suitable route of administration may be employed, including administration by parenteral injection (e.g. , subcutaneous, intramuscular, or intradermal), or by oral, rectal, topical, nasal, ophthalmic, intrathecal, and intracerebral administration.
  • the apoE:NGF and apoE:NT4 complexes are included in an amount effective to accomplish the intended treatment. In general, the claimed apoE complexes are present in an amount effective to enhance survival of neural cells.
  • ApoE NGF and apoE:NT4 complexes may be administered concurrently or in combination with other agents.
  • apoE:NGF and/or apoE:NT4 complexes can be administered with other neurotrophic factors, such as CNTF.
  • an apoE:NGF or apoE:NT4 complex to be administered is determined in a routine manner, and will vary depending on the age and species of subject, the desired effect, the apoE isoform, and the route of administration.
  • Preferred dosages may be determined by simply administering a composition containing a known amount of apoE:NGF or apoE:NT4 complexes in vitro or in vivo to a subject, and monitoring the cells, tissue or subject for the desired effect, as would be known by one skilled in the art.
  • dosages can be as low as 10, 3, 1 , 0.5 or 0.1 ⁇ g/kg body weight, or less. In vivo dosages can be as high as 10, 30, 50 or 100 ⁇ g/kg body weight, or more.
  • the in vivo dosage of apoE:NGF or apoE:NT4 complexes administered will be sufficient to result in peak plasma concentration of the complex of from about lxlO' 1 , 1 x 10° or 1 x 10 1 picomole per Liter to about 1 x 10 ⁇ 1 x 10 3 or even 1 x 10 4 picomole per Liter or more.
  • dosages of the claimed complexes to be administered in vitro there are no particular lower or upper limits to the dosages of the claimed complexes to be administered in vitro.
  • dosages can be as low as 10, 5, 1 , 0.5, or 0.1 ng/ml of medium, or less.
  • In v/tr ⁇ dosages can be as high as 10, 50, 100, 500, 1000, or 1500 ng/ml of medium, or more.
  • apoETFN ⁇ complexes Another aspect of the present invention is apoETFN ⁇ complexes.
  • the biological activity of IFN ⁇ is potentiated (as defined above with respect to NGF and NT4) by complexing with apoE.
  • ApoE: IFN ⁇ complexes can be formed as described above for apoE:NGF and apoE:NT4 complexes.
  • compositions containing the claimed apoETFN ⁇ complexes will generally contain apoE and IFN ⁇ monomers, homodimers and multimers as well. In general, as low as 35% , 25%, 20% , 15% , or lower, or as high as 40% , 50% , 70%, 85% , 90% , 95 %, 99% or higher of the
  • IFN ⁇ molecules in the composition are complexed with apoE.
  • apoETFN ⁇ as used herein encompasses apoETFN ⁇ complexes containing monomers, dimers, trimers and larger multimers of IFN ⁇ . Alternately, apoE monomer, homodimers, homotrimers, etc. may associate with IFN ⁇ monomers, homodimers, homotrimers, etc. Thus, the present invention encompasses complexes between one or more apoE molecules with one or more IFN ⁇ molecules.
  • the active form of IFN ⁇ is a homodimer, and it is believed that the IFN ⁇ homodimer complexes with an apoE monomer. Such heterotrimeric complexes of apoETFN ⁇ are preferred. Those skilled in the art will appreciate that apoE:IFN ⁇ complexes may also be more loosely associated with additional molecules.
  • apoE may first be bound to a substrate prior to being exposed to and forming a complex with IFN ⁇ .
  • the apoETFN ⁇ complex can be bound to a substrate after complex formation.
  • the strength of the binding interaction between apoE and IFN ⁇ is as described above for apoE:NGF and apoE:NT4 complexes.
  • the apoE component of the disclosed complexes is as described above in connection with apoE:NGF and apoE:NT4 complexes.
  • the IFN ⁇ component of the complex can be from a native or recombinant source, and can be produced by any means known in the art.
  • Native U.S. patent No. 5,518,899 to Kurimoto; U.S. Patent No. 4,723,000 to Georgiades et al ; U.S. Patent No. 5,132,110 to Fleischmanri et al.
  • Recombinant U.S. Patent No. 5,602,010 to Hauptmann et al. ; U.S. Patent No. 4,970,161 to Kakutani et al ; U.S. Patent No.
  • the IFN ⁇ component of the claimed apoE:IFN ⁇ complexes can be from any species of origin, preferably of mammalian origin, more preferably of human origin.
  • the term "IFN ⁇ ” also encompasses IFN ⁇ variants, analogs and derivatives, where the variants, analogs and derivatives retain IFN ⁇ biological activity. Exemplary are IFN ⁇ variants, analogs and derivatives disclosed in U.S. Patent No. 4,845,196 to Cowling; PCT application No. 83/04053; U.S. Patent Nos.
  • IFN ⁇ also includes fragments of the IFN ⁇ molecule that bind to IFN ⁇ receptors and/or elicit the biological actions of IFN ⁇ .
  • a further aspect of the present invention is methods of administering compositions containing apoETFN ⁇ complexes to cells in a biologically effective amount.
  • compositions containing apoETFN ⁇ complexes find use in methods of protecting cells against virus infection, such methods comprise administering to the cells an anti-virus effective amount of a composition comprising a complex of apolipoprotein E and IFN ⁇ . See, e.g. , Wheelock (1965) Science 149, 310.
  • protecting cells against viral infection it is meant that virus infection rates are reduced or eliminated or that an already-existing infection is reduced or eliminated by the disclosed methods.
  • Such methods can be used to protect cultured cells in vitro or a subject in vivo.
  • Another aspect of the invention is methods of administering an anti-tumor effective amount of a composition containing apoE:IFN ⁇ complexes.
  • an "anti- tumor effective amount" of the claimed complexes is a dosage that is effective in reducing the incidence of tumor formation or in decreasing the size, growth or metastasis of a tumor.
  • the present invention can be employed to administer the inventive apoETFN ⁇ complexes to treat a subject afflicted with a tumor (e.g. , by decreasing the size, growth or metastasis of a tumor) or to reduce the incidence of tumor formation in a subject at risk of developing tumors.
  • the claimed apoE complexes can be administered to cells in vitro.
  • in vitro administration will simply require adding a solution (i.e. , aqueous) containing the apoE:IFN ⁇ complexes to the culture medium.
  • the apoE and IFN ⁇ components can be added individually, either concurrently or sequentially, to the culture medium.
  • the apoE molecule or the apoE complex can be bound to a substrate (i.e. , a tissue culture dish or petri dish), preferably prior to culturing cells on the substrate. It is well-known in the art that apoE binds to polymeric or coated (i.e. , with extracellular matrix proteins such as laminin) surfaces of tissue culture plates.
  • the disclosed apoE:IFN ⁇ complexes can also be administered to a subject in vivo. Suitable subjects, methods of administration, pharmaceutical formulations of the compositions containing apoETFN ⁇ , and suitable dosages thereof are as described above in connection with apoE:NGF and apoE:NT4 complexes.
  • the disclosed ApoETFN ⁇ complexes can be administered alone or in combination with other therapeutic agents.
  • compositions containing apoE:IFN ⁇ complexes can be administered to a subject in vivo to provide anti-viral and anti-tumor treatments to a subject in need thereof.
  • inventive complexes can also be administered to treat bone degradation and resorption, for example in the elderly, post-menopausal women, and women afflicted with or at risk for developing osteoporosis.
  • IFN ⁇ can be administered to facilitate blood coagulation, by stimulating blood CI inhibitor levels, and to treat type I diabetes mellitus.
  • Complexes of apoE and IFN ⁇ , which potentiate the activity of the IFN ⁇ are useful in vitro for culturing cells that are responsive to IFN ⁇ , including but not limited to bone cells (see U.S. Patent No. 4,921,697 to Peterlik et al).
  • IFN ⁇ is useful in methods of treating ovarian cancer. Also known is use of IFN ⁇ to treat type I diabetes mellitus. U.S. Patent No. 5,624,895 to Sobel. In addition, it has been established that IFN ⁇ is useful in methods of treating blood trauma (U.S. Patent No. 5,271,931 to Lotz et al.) and treating bone degradation (U.S. Patent No. 4,921,697 to Peterlik et al). Thus, the presently claimed IFN ⁇ complexes possessing enhanced biological activity find similar uses as previously known IFN ⁇ compositions.
  • the claimed apoETFN ⁇ complexes also find use in tissue culture, because the complexed IFN ⁇ molecule is protected against proteolytic degradation.
  • the apoETFN ⁇ complexes find use in methods of quantifying or purifying IFN ⁇ receptors or other proteins that bind to IFN ⁇ .
  • ApoE is readily bound to plastic surfaces, such as test tubes and microtiter plates.
  • IFN ⁇ can be tethered to plastic surfaces for use in sandwich assays or affinity purification techniques, which are well-known to those skilled in the art.
  • apoE Delipidated apoE was purified from individuals homozygous for apoE3 or apoE4, as previously described (Rail et al , (1986) Methods Enzymol 128, 273).
  • ApoE3 or apoE4 were incubated with growth factors in Tris-buffered saline (TBS) for up to 4 hours at 37°C. Incubations were terminated by adding 4X SDS-
  • the secondary antibody was a porcine anti-goat IgG conjugated to horseradish peroxidase (HRP; Boehringer Mannheim Biochemicals, Indianapolis, IN) diluted 1:3000 in Blotto.
  • HRP horseradish peroxidase
  • the enzyme-conjugated antibodies were visualized by addition of ECL chemiluminescent substrate (Amersham, Arlington Heights, IL) and exposure to Hyperfilm (Amersham) as previously described (Strittmatter et al , (1993) Proc. Nat. Acad. Sci. USA 90, 1977).
  • Example 2 ADOE3 forms an SDS-Stable Complex with NGF, IFN ⁇ and NT4
  • CNTF human ciliary neurotrophic factor
  • IFN ⁇ neurotrophin-3
  • NT4 neurotrophin-4
  • NGF the fibroblast growth factor bFGF
  • LIF the fibroblast growth factor
  • Sources of growth factors are as follows: CNTF ⁇ generously provided by Regeneron Pharmaceuticals (Tarrytown, NY); IFN ⁇ — Human, recombinant #40044, Collaborative Biomedical Products (Bedford, MA); NT3 - Regeneron Pharmaceuticals; NT4 — human, recombinant #G1511, Promega (Madison, WI); NGF — mouse, natural #40005, Collaborative Biomedical Products (Bedford, MA); bFGF - Collaborative Biomedical Products (Bedford, MA); and LIF - R&D Systems (Minneapolis, MN).
  • CNTF CNTF, IFN ⁇ , NGF and NT4.
  • Complexes were not observed between ApoE3 and IL-6, LIF, NT3, bFGF or BDNF.
  • ApoE4 does not form a SDS-stable complex with any of these growth factors.
  • the gel mobility of each apoE3/growth factor complex is consistent with a bimolecular complex between apoE3 and the growth factor (the active forms of NGF and IFN ⁇ are dimers, so the apoE3/NGF complex is likely a trimer).
  • PC 12 cells require NGF to survive in serum-free medium. Experiments were carried out to assess PC 12 cell survival in the presence of serum-free medium containing NGF, apoE3 or NGF+apoE3.
  • PC 12 cells were plated in 96-well dishes that had been pre-coated with poly- 1-lysine and then treated with a solution containing serial dilutions of NGF and apoE3 that had been preincubated together. It is known that apoE3 will adsorb to tissue culture dishes under these conditions. For the preincubation, serial dilutions of NGF were added to a constant amount of apoE3 (50 ng/ml), incubated for 3 hours at 37°C, and then added to each well. These proteins were allowed to bind to the substrate for an additional 3 hours at 37°C. The final treatments were 50 ng/ml apoE3 + 0, 100 and 1000 ng/ml NGF.
  • apoE3 50 ng/ml of apoE3 mixed with 100 ng/ml NGF and bound to the substrate enhanced survival of PC 12 cells by more than 2- fold as compared with 100 ng/ml soluble NGF alone (p ⁇ 0.001).
  • ApoE alone has no survival promoting activity toward PC 12 cells (even at 1 ⁇ g/ml), regardless of whether the apoE is absorbed to the substrate or included in the culture medium.
  • apoE potentiates the activity of NGF in the maintenance of NGF-responsive peripheral nerve cells.
  • the quantity of formazan product as measured by absorbance at 490nm is proportional to the number of viable cells.
  • 100 ⁇ l of media were removed from each well before adding 20 ⁇ l of MTS solution to each well. Plates were returned to the incubator and absorbance read by a Dynatech MR5000 microplate reader (Dynatech Laboratories) after a four-hour incubation. The data were collected and calculated as the percent difference between each treatment group and the matched control (no apoE/no ⁇ GF condition) for each experiment. The value for the no apoE/no ⁇ GF condition was set at 100% . The percent differences for the separate experiments were then averaged.
  • Example 5 ApoE Potentiates ⁇ GF Activity on Neurite Outgrowth Another well-described activity of NGF is the ability of NGF to promote neurite outgrowth. Studies were carried out in PC 12 cells to evaluate whether apoE would promote NGF activity in this regard. PC 12 cells were first primed for neurite outgrowth by growing cells in 500 ng/ml NGF and serum for 7 days. At this time, all cells possessed neurites greater than 1 cell body in diameter. These neurites were mechanically removed as the cells were replated in 96-well plates. This NGF priming allows for rapid growth of neurites in the experimental condition.
  • phase images of cells were digitally collected. Measurements of neurites were made on random fields using IMAGE 1TM analysis software (Universal Imaging Corp. ; West Chester, PA). ApoE3, but not apoE4, potentiates neurite outgrowth from primed PC 12 cells ( Figure 2). The average neurite outgrowth of cells grown in apoE3 plus NGF was 1.5-fold greater than in cells grown in BSA or apoE4.
  • the nerves were embedded in epoxy resin which was cured at 60°C for 24 hours. Eighty nanometer thin sections were cut on a diamond knife and picked up on formvar covered slot grids which made it possible to view the entire cross-section. Photomicrographs were made on a Phillips 410 electron microscope.
  • DRGs are Impaired on NGF Treated Distal Stumps from ApoE Knockout Mice Neurite outgrowth and Schwann cell migration from neonatal rat dorsal root ganglia (DRGs) were measured on NGF-treated distal stumps from apoE knockout and wildtype mice. For these experiments, neonatal rat DRGs were explanted onto cryostat sections of distal stump from either apoE knockout or wildtype animals. Five days following transection, distal stumps were removed, fresh frozen in O.C.T. compound and cut into 20 ⁇ m thick longitudinal sections. Sections were incubated in 100 ⁇ l of 100 ng/ml NGF for 3 hours at 37°C.
  • Unmyelinated fibers are the fibers that relay pain and temperature sensitivity. Thus, the finding that apoE knockout animals have a substantial decrease in unmylenated fibers (Example 6) suggests that these animals might have a reduced response to noxious thermal stimuli. To test this, the withdrawal latency for wildtype and apoE knockout mice from a hot waterbath was measured.
  • ApoE knockout animals have a 50% loss of unmyelinated, NGF- sensitive, sensory fibers

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Abstract

La présente invention concerne des compositions comprenant des complexes d'apolipoprotéine E et de facteur de croissance du tissu nerveux, de neurotrophine 4 ou d'interféron Ω. L'apolipoprotéine E peut présenter n'importe quelle isoforme, mais elle sera de préférence constituée par l'apolipoprotéine E3. Les complexes préférés sont des complexes covalents d'apolipoprotéine E et de facteur de croissance du tissu nerveux, d'interféron Ω ou de neurotrophine 4. L'invention se rapporte en outre à des procédés permettant d'augmenter la survie de cellules neuronales en administrant une composition comprenant un complexe d'apolipoprotéine E et de facteur de croissance du tissu nerveux ou de neurotrophine 4. L'invention concerne aussi des procédés permettant d'administrer des compositions comprenant des complexes d'apolipoprotéine E et d'interféron Ω dans le but de protéger des infections virales, de traiter le diabète sucré, l'ostéolyse, les lésions sanguines, et de produire des effets anti-tumoraux. Les procédés de la présente invention peuvent être utilisés in vitro et in vivo.
EP98950816A 1997-09-30 1998-09-29 Complexes d'apolipoproteine e/facteur de croissance et procedes d'utilisation Withdrawn EP1019081A2 (fr)

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WO2017165515A1 (fr) * 2016-03-23 2017-09-28 The Research Institute At Nationwide Children's Hospital Stimulation de la croissance osseuse à l'aide d'apolipoprotéine e

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US5604202A (en) * 1990-11-13 1997-02-18 Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University Use of NGF growth factors to treat drug-induced neuropathy
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