Compositions with nerve regeneration applications

Abstract

Disclosed herein is a pharmaceutical composition containing transferrin and / or lactoferrin for use in promoting and / or inducing the generation of new nerve cells in patients suffering from neurodegenerative events resulting from at least one of traumatic brain injury, non-traumatic brain injury, spinal cord injury, peripheral nerve injury, or peripheral neuropathy. Ideally, the transferrin and / or lactoferrin has low iron saturation.

Description

[Technical Field]

[0001] The present invention relates to therapeutic proteins and their use in the field of regenerative medicine. In particular, disclosed herein is the application of transferrin and lactoferrin and their use in promoting the proliferation, induction, and / or differentiation of neural progenitor or neural stem cells in patients suffering from nerve injury. [Background technology]

[0002] Injuries to the brain and spinal cord can manifest as immediate or chronic neurodegenerative effects that devastatingly alter the quality of life of affected individuals. Acquired brain injury (ABI), i.e., injuries that are not hereditary, congenital, or induced at birth, results in changes in the brain's neural activity that affect the physical integrity, metabolic activity, or functional capacity of neurons in the brain. There are two types of acquired brain injury: traumatic and non-traumatic.

[0003] Traumatic brain injury (TBI) is characterized by altered brain function or other evidence of brain pathology caused by an external force. Traumatic impact injuries can be defined as non-penetrating or penetrating and include falls, assaults, motor vehicle accidents, and sports injuries. Non-traumatic brain injury (NBTI) causes damage to the brain through non-impact processes, such as lack of oxygen, exposure to toxins, or pressure from tumors. Examples of NTBI include lack of oxygen supply to the brain caused by stroke, aneurysm, and heart attack.

[0004] Stroke, one of the most common categories of NBTI, occurs when brain tissue is deprived of oxygen and nutrients due to an interruption or reduction in blood supply to a part of the brain. The neurodegenerative process begins almost immediately, and brain cells begin to die within minutes. Ischemic stroke occurs when a blood vessel (artery) supplying blood to an area of ​​the brain becomes blocked by a blood clot. Hemorrhagic stroke occurs when an artery in the brain leaks or ruptures. Of the two, ischemic stroke is the most common form of stroke.

[0005] The prevalence of stroke in modern society creates an enormous burden on healthcare infrastructure and costs. Currently, the only therapeutic agent approved by the FDA for ischemic stroke is recombinant tissue plasminogen activator (rtPA). The primary function of rtPA is to dissolve blood clots and promote reperfusion. An alternative method for reestablishing obstructed blood flow is through surgical intervention.

[0006] rtPA treatment has a narrow therapeutic window, thus limiting its widespread application. Furthermore, while restoring perfusion to ischemic tissues by rtPA is important, the cascade of necrosis, apoptosis, and inflammation begins within minutes of severe oxygen deprivation. There is growing speculation that genetically programmed neuronal cell death during post-ischemic tissue inflammation (which can last from days to weeks) significantly contributes to the eventual pathology due to delays in patient treatment / evaluation. Thus, permanent neuronal and neuronal damage may occur even before the patient is evaluated.

[0007] At the time of writing, there are no approved neuroregenerative therapies that can reverse the effects of neurodegeneration associated with TBI and NTBI. Given the lack of curative therapies, it is not surprising that the majority of literature in this field is biased toward neuroprotection, a non-restorative approach to mitigating neuronal cell death by pre- or co-administration of specific molecules in response to anticipated neurodegenerative ischemia or reperfusion events.

[0008] One such example is U.S. Patent Publication No. 2016008437 in the name of Grifols Worldwide Operations Ltd, which discloses apo-transferrin as exerting neuroprotective effects by modulating the activity of hypoxia-inducible factor (HIF) in a rat model of stroke. The inventors observed the neuroprotective effect of apo-transferrin manifested as a reduction in the volume of the infarcted area in rats treated with apo-transferrin compared to control rats. [Prior art documents] [License]

[0009] [License 1] U.S. Patent and Trademark Office No. 2016008437 [Non-licensed literature]

[0010] [Non-licensed Document 1] Ceriotti, Improved direct specific determination of serum iron and total iron-binding capacity Clin Chem. 1980, 26(2), pages 327~31 [Non-licensed Document 2] Manley, Simultaneous Cu-, Fe-, and Zn-specific detection of metalloproteins contained in rabbit plasma by size-exclusion chromatography-inductively coupled plasma atomic emission spectroscopy. J Biol Inorg Chem. 2009, 14, 61~74 pages [Non-licensed Document 3] L von Bonsdorff, Transferrin, Chapter 21, Pages 301~310, Production of Plasma Proteins for Therapeutic Use, edited by J. Bertolini, Wiley, 2013 [Print ISBN: 9780470924310 オンラインISBN: 9781118356807] [Non-licensed Document 4] Arvidsson, 2002, Nat. Med., 8, pages 963~970 [Non-licensed Document 5] Kokaia and Lindvall, 2003, Curr. Opin. Neurobiol., 13, pages 127~132 [Non-licensed Document 6] Kernie et al., 2010, Neurobiol. Disease, 37, 267-274 [Non-Patent Document 7] Remington: The Science and Practice of Pharmacy, 21st ed., 2005, edited by D.B. Troy, Lippincott Williams & Wilkins, Philadelphia [Non-patent document 8] Encyclopedia of Pharmaceutical Technology, edited by J. Swarbrick and J.C. Boylan, 1988-1999, Marcel Dekker, New York [Non-Patent Document 9] Pfeiffer et al., Am J Clin Nutr 2017, 106(Suppl), 1606S-14S [Non-Patent Document 10] Agholme, 2010. J. of Alzheimer's Disease. Vol. 20:1p069~108; [Non-Patent Document 11] Dyberg et al., 2017. PNAS Volume 114(32), E6603-E6612 [Non-Patent Document 12] Dayem et al. Biologically synthesized silver nanoparticles induce neuronal differentiation of SH-SY5Y cells via modulation of reactive oxygen species, phosphatases, and kinase signaling pathways. Biotechnol. J. 2014, 9, pp. 934-943. [Non-Patent Document 13] Fagerstrom et al. Protein Kinase C-epsilon Implicated in Neurite Outgrowth in Differentiating Human Neuroblastoma Cells. Cell Growth & Differentiation, Vol. 7, pp. 775-785, June 1996. [Non-Patent Document 14] Han et al. Berberine. Promotes Axonal Regeneration in Injured Nerves of the Peripheral Nervous System. J Med Food 15 (4) 2012, pp. 413-417. [Non-Patent Document 15] Gold et al. Nonimmunosuppressant FKBP-12 Ligand Increases Nerve Regeneration. EXPERIMENTAL NEUROLOGY 147, pp. 269-278 (1997). [Non-Patent Document 16] Kim et al. Protective effect of GCSB-5, an herbal preparation, against peripheral nerve injury in rats. Journal of Ethnopharmacology 136 (2011) pp. 297-304. [Non-Patent Document 17] Lesma et al. Glycosaminoglycans in Nerve Injury: I. Low Doses of Glycosaminoglycans Promote Neurite Formation. Journal of Neuroscience Research. 1996 46(5):565-71. [Non-Patent Document 18] Hattangady and Rajadhyaksha. A brief review of in vitro models of diabetic neuropathy. Int J Diabetes Dev Ctries. 2009 Oct-Dec;29(4):143-149. [Non-Patent Document 19] Vincent et al. Oxidative Stress and Programmed Cell Death in Diabetic Neuropathy. Ann. NY Acad. Sci. 959: 368-383 (2002). [Non-Patent Document 20] Shindo. Modulation of Basal Nitric Oxide-dependent Cyclic-GMP Production by Ambient Glucose, Myo-Inositol, and Protein Kinase C in SH-SY5Y Human Neuroblastoma Cells. J. Clin. Invest. Vol. 97, No. 3, February 1996, pp. 736-745. [Non-Patent Document 21] Li et al. C-peptide enhances insulin-mediated cell growth and protection against high glucose-induced apoptosis in SH-SY5Y cells. Diabetes Metab Res Rev 2003; 19: pp. 375-385. [Non-Patent Document 22] Rigolio et al. Resveratrol interference with the cell cycle protects human neuroblastoma SH-SY5Y cell from paclitaxel-induced apoptosis. Neurochemistry International 46 (2005) pp. 205-211. [Non-Patent Document 23] Donzelli et al. Neurotoxicity of platinum compounds: comparison of the effects of cisplatin and oxaliplatin on the human neuroblastoma cell line SH-SY5Y. Journal of Neuro-Oncology 67: pp. 65-73, 2004. [Non-Patent Document 24] Mannelli et al. Oxaliplatin-induced oxidative stress innervous system-derived cellular models: Could it correlate with in vivo neuropathy? Free Radical Biology and Medicine 61 (2013) pp. 143-150. [Non-Patent Document 25] Hong et al. Neurotoxicity induced in differentiated SK-N-SH-SY5Y human neuroblastoma cells by organophosphorus compounds. Toxicology and Applied Pharmacology 186 (2003) pp. 110-118. [Non-Patent Document 26] Ehrich et al. Interaction of organophosphorus compounds with muscarinic receptors in SH-SY5Y human neuroblastoma cells. Journal of Toxicology and Environmental Health 1994 43(1):51-63. [Non-Patent Document 27] Triyoso and Good. Pulsatile shear stress leads to DNA fragmentation in human SH-SY5Y neuroblastoma cell line. Journal of Physiology (1999), 515.2, pp. 355-365. [Non-patent document 28] Song et al. Arctigenin Confers Neuroprotection Against Mechanical Trauma Injury in Human Neuroblastoma SH-SY5Y Cells by Regulating miRNA-16 and miRNA-199a Expression to Alleviate Inflammation. J Mol Neurosci (2016) 60:115–129. [Non-Patent Document 29] Skotak et al. An in vitro injury model for SH-SY5Y neuroblastoma cells: Effect of strain and strain rate. Journal of Neuroscience Methods 205 (2012) pp. 159-168. [Non-Patent Document 30] Arun et al. Studies on blast traumatic brain injury using in-vitro model with shock tube. NeuroReport (2011) 22:379-384. [Non-Patent Document 31] Miglio et al. Cabergoline protects SH-SY5Y neuronal cells in an in vitro model of ischemia. European Journal of Pharmacology 489 (2004) pp. 157-165. [Non-Patent Document 32] Duong et al. Multiple protective activities of neuroglobin in cultured neuronal cells exposed to hypoxia re-oxygenation injury. J. Neurochem. (2009) 108, pp. 1143-1154. [Non-Patent Document 33] Qiu et al. Enhancement of ischemia-induced tyrosine phosphorylation of Kv1.2 by vascular endothelial growth factor via activation of phosphatidylinositol 3-kinase. J. Neurochem. (2003) 10.104. [Non-Patent Document 34] Azari and Reynolds, “In Vitro Models for neurogenesis”. Cold Spring Harb Perspect Biol 2016, 8, a021279 [Non-Patent Document 35] Silva et al., 2009. Biochimica et Biophysica Acta, vol. 1794, pp. 1449-1458 [Non-Patent Document 36] Chowdhury et al., 2013. ACS Chem. Biol. Vol. 8, p. 1488 [Non-Patent Document 37] Houlton et al., 2019. Frontiers in Neurosci., Volume 13, Paper 790 [Non-Patent Document 38] Weissmiller and Wu, 2012. Translational Neurodegeneration, Vol. 1:14 [Non-Patent Document 39] Apfel, 2001. Clin Chem Lab Med., vol. 39(4), p. 351 [Non-Patent Document 40] Anglada-Huguet et al., 2014, Molecular Neurobiology, Vol. 49, pp. 784-795 [Non-Patent Document 41] Denninger et al., 2018, J. Vis. Exp., Vol. 141, e58593 Summary of the Invention [Problem to be solved by the invention]

[0011] Notwithstanding the above, there are clearly insufficient clinical candidates with the potential to reverse the debilitating effects of neurodegeneration associated with spinal cord injury, TBI, and NBTI. Therapies that address this need and at least partially reverse traumatic and non-traumatic neuronal injury remain elusive and therefore highly desirable. [Means for solving the problem]

[0012] The words "comprise" and "having" when used herein in connection with the present invention are used to specify the presence of stated features, integers, steps or components, but do not exclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0013] Those skilled in the art will understand that the specific embodiments disclosed herein should not be read in isolation, and that the specification intends for the disclosed embodiments to be read in combination with one another, rather than individually. As such, each embodiment may serve as a basis for modifying or limiting other embodiments disclosed herein.

[0014] Concentrations, amounts, and other numerical data may be expressed or presented herein in range format. It should be understood that such range format is used merely for convenience and brevity and, thus, should be interpreted flexibly to include not only the numerical values ​​specified as the limits of the range, but also all individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were expressly stated. By way of example, a numerical range of "10 to 100" should be interpreted not only to include the specified value of 10 to 100, but also to include each individual value and subrange within the stated range. Thus, included within this numerical range are individual values ​​such as 10, 11, 12, 13... 97, 98, 99, 100, etc., as well as subranges such as 10 to 40, 25 to 40, and 50 to 60. This same principle applies to ranges reciting only a single numerical value, such as "at least 10." Furthermore, such interpretation shall apply regardless of the breadth of the range or the characteristic being recited.

[0015] Treatment method In a first aspect, the present invention provides a method of promoting and / or inducing the generation of new nerve cells in a patient suffering from a neurodegenerative event resulting from at least one of traumatic brain injury, non-traumatic brain injury, spinal cord injury, peripheral nerve injury, or peripheral neuropathy, comprising: The method includes administering to a patient in need thereof a therapeutically effective amount of a protein selected from transferrin, lactoferrin, and combinations thereof.

[0016] In one embodiment, the patient may be suffering from a neurodegenerative event resulting from at least one of stroke, peripheral nerve injury, traumatic brain injury, or peripheral neuropathy. For example, the patient may be suffering from a neurodegenerative event resulting from peripheral nerve injury. In one embodiment, the patient may be suffering from a neurodegenerative event resulting from stroke, for example, ischemic stroke or hemorrhagic stroke. In one embodiment, the patient may be suffering from a neurodegenerative event resulting from ischemic stroke.

[0017] Those skilled in the art will recognize that, among the numerous mammalian iron-binding proteins, transferrin and lactoferrin are related proteins of the transferrin family, sharing 61% sequence identity. In addition to some overlapping and complementary functions, transferrin and lactoferrin also exhibit some mutually exclusive functions. The present invention includes within its scope all wild-type mammalian transferrin proteins, although human transferrin (UniProtKB SEQ ID NO: Q06AH7) comprising the amino acid sequence set forth in SEQ ID NO: 1 is particularly preferred. Similarly, the present invention includes within its scope all wild-type mammalian lactoferrin proteins, although human lactoferrin (UniProtKB SEQ ID NO: P02788) comprising the amino acid sequence set forth in SEQ ID NO: 2 is particularly preferred.

[0018] Wild-type transferrin protein contains two homologous lobes (N- and C-lobes), each of which binds a single iron atom. Thus, each wild-type transferrin molecule can bind up to two iron atoms or ions per molecule. Similarly, each wild-type lactoferrin molecule can bind two iron atoms per molecule in a similar manner.

[0019] Transferrin and lactoferrin can be extracted from natural sources or alternatively produced using recombinant production / manufacturing processes. Suitable natural sources can be human plasma or human milk, respectively.

[0020] "Transferrin" as used herein refers to a therapeutically effective amount of: wild-type (mammalian, preferably human) transferrin protein, its functional variants, a functional fragment thereof, or combinations thereof is interpreted to mean

[0021] The iron saturation of transferrin, a functional variant thereof, or a functional fragment thereof may be about 50% or less. Preferably, the iron saturation is about 40% or less. In one embodiment, the iron saturation is about 30% or less. For example, the iron saturation may be about 20% or less, e.g., about 10% or less. In some embodiments, the iron saturation is about 5% or less. In still further embodiments, the iron saturation may be less than about 1%. For the avoidance of doubt, a range presented herein as less than X% includes 0 to X%, i.e., transferrin with no iron bound thereto, i.e., 0% iron saturation.

[0022] As used herein, "apo-transferrin" shall mean transferrin with less than 1% iron saturation. Similarly, "holo-transferrin" shall mean transferrin with 99% or greater iron saturation.

[0023] Those skilled in the art will appreciate that the iron saturation level of transferrin can be readily determined without undue burden by quantifying the total iron level in a sample having a known transferrin concentration. The total iron level in a sample can be measured by any one of several methods known in the art.

[0024] Suitable examples include: Colorimetric assay - Iron is detected by ferrozine and Fe in acetate buffer. 2+ The intensity of the purple complex formed in the reaction between thiourea or other chemicals is quantified by measuring at 562 nm. 2+ Iron-binding capacity is sometimes increased by adding ferrozine to form complexes with contaminating metals such as iron, which can also bind to ferrozine, resulting in falsely elevated iron levels. See Ceriotti et al., Improved direct specific determination of serum iron and total iron-binding capacity, Clin Chem. 1980, 26(2), 327-31, the contents of which are incorporated herein by reference. Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) - an emission spectroscopy method that quantifies the mass percentage of metals in a sample. ICP-AES is based on using plasma (an ionized gas consisting of positive ions and free electrons) to excite metal atoms / ions in a sample and then analyzing the emission wavelength of electromagnetic radiation typical of that particular metal. While this technique is a standard analytical method within the general knowledge of those skilled in the art, more information on ICP-AES can be found in Manley et al., "Simultaneous Cu-, Fe-, and Zn-specific detection of metalloproteins contained in rabbit plasma by size-exclusion chromatography-inductively coupled plasma atomic emission spectroscopy." J Biol Inorg Chem. 2009, 14, pp. 61-74 (the contents of which are incorporated herein by reference).

[0025] The preferred method for determining the iron content of a sample for the therapeutic methods of the present invention is ICP-AES. The iron saturation of transferrin is then calculated based on the transferrin protein concentration, the total iron content of the sample, and the fact that wild-type transferrin has two iron-binding sites. Because wild-type human transferrin (molecular weight 79,750) can bind two iron atoms, a sample containing 1 g of transferrin would be 100% saturated with 1.4 mg of iron.

[0026] If the transferrin concentration of a particular sample is not known, it can be readily determined by a variety of well-characterized immunological (ELISA, turbidimetric) and non-immunological methods (absorbance, AU480 chemistry assay).

[0027] At the time of writing, transferrin has not been approved as a drug in any major jurisdiction in the world. Therefore, there is no pharmacopoeial monograph for transferrin. Further information about the physical properties of transferrin, such as iron saturation, can be obtained from major reference books consulted by those skilled in the art. See L. von Bonsdorff et al., Transferrin, Chapter 21, pp. 301-310; Production of Plasma Proteins for Therapeutic Use, edited by J. Bertolini et al., Wiley, 2013 [print ISBN: 9780470924310 online ISBN: 9781118356807], the contents of which are incorporated herein by reference and deemed to be within the general knowledge of those skilled in the art.

[0028] "Lactoferrin" as used herein refers to a therapeutically effective amount of: wild-type (mammalian, preferably human) lactoferrin protein, its functional variants, a functional fragment thereof, or combinations thereof is interpreted to mean

[0029] The iron saturation of lactoferrin, its functional variant, or its functional fragment may be about 50% or less. Preferably, the iron saturation is about 40% or less. In one embodiment, the iron saturation is about 30% or less. For example, the iron saturation may be about 20% or less, e.g., about 10% or less. In some embodiments, the iron saturation is about 5% or less. In still further embodiments, the iron saturation may be less than about 1%.

[0030] As used herein, "apo-lactoferrin" refers to lactoferrin with an iron saturation of less than 1%. Similarly, "holo-lactoferrin" refers to lactoferrin with an iron saturation of 99% or more. The iron content and saturation level of lactoferrin can be measured similarly to that of transferrin, as discussed in detail above.

[0031] In using the terms transferrin and lactoferrin, the present specification includes within its scope recombinant derivatives of transferrin and lactoferrin that differ from the wild-type amino acid sequences of the human proteins outlined in SEQ ID NOs: 1 and 2, respectively, by one or more substitutions, one or more deletions, or one or more insertions that are unlikely to substantially alter the structure or hydropathic index properties of the recombinant proteins relative to the wild-type proteins. Recombinant variants of transferrin and lactoferrin within the scope of the present invention may additionally comprise at least one post-translational modification, such as PEGylation, glycosylation, polysialylation, or a combination thereof.

[0032] In one embodiment, the present invention contemplates recombinant variants of transferrin and lactoferrin having one or more conservative substitutions relative to the wild-type proteins in SEQ ID NOs: 1 and 2. A "conservative substitution" is one in which an amino acid is substituted with another amino acid of similar properties, such that one skilled in the art of peptide chemistry would predict that the secondary structure and hydropathic index properties of the polypeptide will not be substantially altered. Generally, changes within the following groups of amino acids represent conservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

[0033] For example, recombinant transferrin or lactoferrin within the scope of the therapeutic methods of the present invention may have at least 90%, 95%, 96%, 97%, 98%, or 99% homology to the wild-type human transferrin and human lactoferrin proteins outlined in SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0034] In further embodiments, the present invention includes specific mutant forms of transferrin and / or lactoferrin that maintain their structure but do not allow the proteins to bind iron in the iron-binding domains, e.g., the N-lobe, one or other of the C-lobe, or a combination thereof.

[0035] Transferrin variants within the scope of the present invention include, but are not limited to: i) Y188F mutant N-lobe (SEQ ID NO: 3); ii) Y95F / Y188F mutant N-lobe (SEQ ID NO: 4); and iii) Y426F / Y517F mutant C-lobe (SEQ ID NO: 5).

[0036] Those skilled in the art will appreciate that recombinant proteins can be obtained using standard techniques well known in the art of protein expression, production, and purification. The nucleic acid sequence of the recombinant protein of interest can be inserted into any expression vector suitable for expression in the host cell of choice, such as mammalian cells, insect cells, plant cells, yeast, and bacteria.

[0037] As used herein, the term "expression vector" refers to an entity capable of introducing a protein expression construct into a host cell. Some expression vectors also replicate within the host cell, thereby increasing protein expression by the protein expression construct. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Other vectors include cosmids, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs), fosmids, phages, and phagemids. Another type of vector is a viral vector, into which additional DNA segments can be ligated. Some vectors are capable of autonomous replication within host cells into which they are introduced (e.g., vectors with an origin of replication that functions within the host cell). Other vectors can integrate into the genome of a host cell when introduced into the host cell, thereby replicating along with the host genome. Furthermore, certain preferred vectors are capable of directing the expression of genes to which they are operably linked.

[0038] Suitable bacterial cells include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, Pseudomonas spp., Streptomyces spp., and Staphylococcus spp. Suitable yeast cells include Saccharomyces spp., Pichia spp., and Kluyveromyces spp. Suitable insect cells include those derived from the silkworm (Bombyx mori), the armyworm (Mamestra brassicae), the Spodoptera frugiperda, the nettle looper (Trichoplusia ni), and the fruit fly (Drosophila melanogaster). Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, COS, MDCK, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0, CRL7O3O, HsS78Bst, human hepatocellular carcinoma cells (e.g., Hep G2), human adenovirus-transformed 293 cells (e.g., HEK293), PER.C6, murine L-929 cells, HaK hamster cell line, murine 3T3 cells derived from Swiss, Balb-c, or NIH mice, and CV-1 cell line cells.

[0039] The present invention also contemplates the use of wild-type and recombinant transferrin and lactoferrin proteins conjugated or fused to any other protein, protein fragment, protein domain, peptide, small molecule, or other chemical entity. For example, suitable fusion or conjugation partners include serum albumin (e.g., bovine, rabbit, or human), keyhole limpet hemocyanin, immunoglobulin molecules (including immunoglobulin Fc domains), thyroglobulin, ovalbumin, tetanus toxoid or toxoids from other pathogenic bacteria, or attenuated toxin derivatives, cytokines, chemokines, glucagon-like peptide-1, exendin-4, XTEN, or combinations thereof.

[0040] In one embodiment of the invention, the transferrin and lactoferrin proteins used in the methods of the invention are fusion proteins with improved in vivo half-lives, where: a wild-type (mammalian, preferably human) transferrin or lactoferrin protein fused to a fusion partner selected from an immunoglobulin Fc domain and albumin; or The variant transferrin or lactoferrin protein within the method of the present invention is fused to a fusion partner selected from an immunoglobulin Fc domain and albumin.

[0041] In one embodiment, the preferred fusion partner is an immunoglobulin Fc domain. For example, the immunoglobulin Fc domain may comprise at least a portion of a constant heavy chain immunoglobulin domain. The constant heavy chain immunoglobulin domain is preferably an Fc fragment comprising the CH2 and CH3 domains and optionally at least a portion of the hinge region. The immunoglobulin Fc domain may be an IgG, IgM, IgD, IgA, or IgE immunoglobulin Fc domain, or a modified immunoglobulin Fc domain derived therefrom. Preferably, the immunoglobulin Fc domain comprises at least a portion of a constant IgG immunoglobulin Fc domain. The IgG immunoglobulin Fc domain may be selected from an IgG1, IgG2, IgG3, or IgG4 Fc domain, or a modified Fc domain thereof.

[0042] In one embodiment, the fusion protein may comprise transferrin fused to an IgG1 Fc domain. In one embodiment, the fusion protein may comprise a transferrin mutant fused to an IgG1 Fc domain.

[0043] Neurodegenerative events Surprisingly, the inventors have discovered that both transferrin and lactoferrin have unexpected therapeutic roles beyond iron binding / delivery to cells, in that they were highly effective in stimulating neuronal development from neural progenitor cells and / or neural stem cells. Accordingly, the present invention provides a method for stimulating neuronal development in a patient suffering from a neurodegenerative event resulting from at least one of traumatic brain injury, non-traumatic brain injury, spinal cord injury, peripheral nerve injury, or peripheral neuropathy, comprising: The method includes administering to a patient in need thereof a therapeutically effective amount of a protein selected from transferrin, lactoferrin, and combinations thereof.

[0044] As used herein, the term "stimulating neural cell development" is used to mean that transferrin or lactoferrin has a direct or indirect effect on neural progenitor cells and / or neural stem cells in a patient to produce new neural cells. Without wishing to limit the generality of the invention, administration of transferrin or lactoferrin may result in an increase in neural progenitor / neural stem cells compared to neural progenitor / neural stem cells that have not been exposed to transferrin or lactoferrin. i) the proliferation of neural progenitor cells and / or neural stem cells in a patient; or ii) Inducing differentiation of neural progenitor cells and / or neural stem cells into differentiated neural cells. It is expected that at least one of the following will increase as a result.

[0045] "Neuronal cells," as used herein, include all cells of the nervous system, including, but not limited to, glial cells and neurons. In one embodiment, the neuronal cells referred to in the methods of the present invention are neuronal cells, and transferrin and lactoferrin enhance neurogenesis of new neurons.

[0046] As used herein, the term "neurodegenerative event" refers to an event that causes loss of neuronal structure and / or function and includes neuronal death. The event may be an isolated, single event / occurrence that causes immediate neuronal damage or death. Alternatively, the event may be a continuous or chronic event that gradually leads to increasing levels of neuronal damage or death. In certain embodiments, a neurodegenerative event causes loss of structure, loss of function, or neuronal (or neuron) death in the brain and / or spinal cord, resulting in damage and dysfunction in the brain and / or spinal cord.

[0047] In the context of the methods of the present invention, the neurodegenerative event results from at least one of traumatic brain injury, non-traumatic brain injury, spinal cord injury, peripheral nerve injury, or peripheral neuropathy.

[0048] As used herein, the term "traumatic brain injury" refers to damage to the brain caused by penetrating or non-penetrating trauma to the head. There are many possible causes, non-limiting examples of which include road traffic accidents, assaults, sports collisions, unprotected falls, etc.

[0049] As used herein, the term "non-traumatic brain injury" refers to damage to the brain resulting from a non-traumatic cause. Suitable non-limiting examples of non-traumatic causes include tumors, stroke, transient ischemic attacks, cerebral hemorrhage, hemorrhagic stroke, toxins / drugs, hypoxic encephalopathy, anoxic encephalopathy, ingestion of chemical toxins, hydrocephalus, meningitis, and encephalitis. In one embodiment, the non-traumatic brain injury is caused by a stroke, for example, an ischemic stroke or a hemorrhagic stroke. For example, the non-traumatic brain injury may be caused by an ischemic stroke.

[0050] Similarly, "spinal cord injury" is herein interpreted to mean damage to any portion of the spinal cord or nerves at the end of the spinal canal that results in loss of function, such as mobility and / or sensation. Non-limiting causes of spinal cord injury include trauma (car accidents, gunshots, falls, etc.), disease (polio, spina bifida, etc.), infection, and tumors.

[0051] In one embodiment, the patient may be suffering from a neurodegenerative event resulting from at least one of stroke, peripheral nerve injury, traumatic brain injury, or peripheral neuropathy. For example, the patient may be suffering from a neurodegenerative event resulting from peripheral nerve injury. In one embodiment, the patient may be suffering from a neurodegenerative event resulting from stroke, for example, ischemic stroke or hemorrhagic stroke. In one embodiment, the patient may be suffering from a neurodegenerative event resulting from ischemic stroke.

[0052] As a non-limiting / non-binding theory, it is known that neurodegenerative injury or damage induces neural stem cells to migrate to the site of such injury or damage. See Arvidsson et al., 2002, Nat. Med., 8, 963-970; Kokaia and Lindvall, 2003, Curr. Opin. Neurobiol., 13, 127-132; and Kernie et al., 2010, Neurobiol. Disease, 37, 267-274. The present inventors speculate that by increasing the concentration of transferrin, lactoferrin, or a combination thereof in a patient, such molecules may enhance and / or promote the body's own nerve regeneration and repair mechanisms. Transferrin and lactoferrin can be administered directly or indirectly to the site of neurodegenerative injury or damage by any conventional drug delivery means known to those skilled in the art.

[0053] Those skilled in the art will understand that the specific embodiments disclosed in the above paragraphs should not be read in isolation, and that the specification intends for these embodiments to be disclosed in combination with other embodiments, not individually. For example, each of the embodiments disclosed in the above paragraphs should be read as expressly combined with each of the embodiments in the above paragraphs, or with two or more of the embodiments disclosed therein, in any order.

[0054] Combination therapy The methods of the present invention also contemplate the use of supplementary active compounds and molecules in combination with transferrin and / or lactoferrin. The supplementary active compounds and molecules can be formulated with transferrin or lactoferrin as unit dosage forms, i.e., as physically separate units intended as a unitary dosage for the subject to be treated. Alternatively, the supplementary active compounds and molecules can be provided as a kit of parts, comprising: administered separately from transferrin and / or lactoferrin in a stepwise or sequential dosing pattern; or · Co-administered simultaneously from different dosage forms.

[0055] For example, the methods of the present invention contemplate administering other serum- or plasma-based proteins in combination with transferrin and / or lactoferrin. Serum or plasma proteins within the scope of the present invention include those purified from a suitable plasma source, such as human plasma, and those prepared using recombinant manufacturing techniques. For example, the serum or plasma protein may be selected from the group consisting of albumin (e.g., albutein), alpha-1 antitrypsin / alpha-1 proteinase inhibitor (e.g., prolastin), antithrombin (e.g., thrombate III), polyclonal immunoglobulins (IgG, IgA, and combinations thereof), polyspecific immunoglobulin (IgM), C1 esterase inhibitor (e.g., berinex), transthyretin, and combinations thereof.

[0056] Exemplary polyclonal immunoglobulins within the scope of the present invention include commercially available polyclonal IgG preparations such as FLEBOGAMMA DIF 5% and 10%, GAMUNEX-C 10%, BIVIGAM 10%, GAMMAGARD Liquid 10%, and the like.

[0057] Exemplary polyspecific immunoglobulins (IgM) within the scope of the present invention include commercially available immunoglobulin preparations containing polyspecific IgM, such as PENTAGLOBIN or TRIMODULIN.

[0058] In one embodiment, the serum or plasma protein may be selected from the group consisting of albumin, antithrombin, alpha-1 antitrypsin, C1 esterase inhibitor, and combinations thereof. For example, the serum or plasma protein may be selected from the group consisting of antithrombin, alpha-1 antitrypsin, and combinations thereof. In certain embodiments, a therapeutically effective amount of alpha-1 antitrypsin is administered to the patient in addition to a protein selected from transferrin, lactoferrin, and combinations thereof. In certain embodiments, a therapeutically effective amount of antithrombin is administered to the patient in addition to a protein selected from transferrin, lactoferrin, and combinations thereof.

[0059] The methods of the present invention also provide for administering known neurogenic / neurotrophic compounds and molecules in combination with transferrin and / or lactoferrin. For example, the methods of the present invention contemplate administering neurogenic / neurotrophic proteins, peptides, and small molecules in conjunction with transferrin and / or lactoferrin.

[0060] Suitable neurogenic and / or neurotrophic compounds and molecules may be selected from the group consisting of BDNF (brain-derived neurotrophic factor; NGF superfamily; SEQ ID NO: 6), GNDF (glial cell line-derived neurotrophic factor; TGF-β superfamily; SEQ ID NO: 7), CNTF (cilliary neurotrophic factor-1; neurokine superfamily; SEQ ID NO: 8), PACAP (amino acids 1-38 of pituitary adenylate cyclase-activating polypeptide; SEQ ID NO: 9), Y-27632 and pharmaceutically acceptable salts thereof [trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide], fasudil and pharmaceutically acceptable salts thereof [hexahydro-1-(5-isoquinolinyl-sulfonyl)-1H-1,4-diazepine], and combinations thereof.

[0061] Those skilled in the art will appreciate that the present invention also contemplates within its scope covalent conjugates of each of the above-listed compounds and molecules to transferrin and lactoferrin, respectively. Additionally, the present invention will also contemplate recombinant fusion proteins of each of the above-listed proteins and peptides with transferrin and lactoferrin, respectively.

[0062] In one embodiment, transferrin, lactoferrin, or a combination thereof may comprise at least 20% by weight of the total protein content utilized in the therapeutic methods of the invention. For example, transferrin, lactoferrin, or a combination thereof may comprise about 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99% or more by weight of the total protein content utilized in the combination therapy of the invention.

[0063] Those skilled in the art will understand that the specific embodiments disclosed in the above paragraphs should not be read in isolation, and that the specification intends for these embodiments to be disclosed in combination with other embodiments, not individually. For example, each of the embodiments disclosed in the above paragraphs should be read as expressly combined with each of the embodiments in the above paragraphs, or with two or more of the embodiments disclosed therein, in any order.

[0064] Pharmaceutical compositions of the present invention In a further aspect, the present invention also provides pharmaceutical compositions comprising transferrin, lactoferrin, or a combination thereof for use in generating new nerve cells in patients suffering from neurodegenerative events resulting from traumatic brain injury, non-traumatic brain injury, spinal cord injury, and combinations thereof.

[0065] The pharmaceutical composition of the present invention may optionally further comprise at least one pharmaceutically acceptable carrier. The at least one pharmaceutically acceptable carrier may be selected from adjuvants and vehicles. The at least one pharmaceutically acceptable carrier includes any and all solvents, diluents, other liquid vehicles, dispersion aids, suspension aids, surfactants, tonicity adjusters, thickeners, emulsifiers, and preservatives suitable for the desired specific dosage form.

[0066] Suitable carriers are described in Remington: The Science and Practice of Pharmacy, 21st ed., 2005, edited by DB Troy, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, edited by J. Swarbrick and JC Boylan, 1988-1999, Marcel Dekker, New York (the contents of which are incorporated herein by reference). Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, glycol, dextrose solution, buffer solutions (such as phosphate, glycine, sorbic acid, and potassium sorbate), and 5% human serum albumin. Liposomes and non-aqueous vehicles, such as glyceride mixtures of saturated vegetable fatty acids and fixed oils (such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil), may also be used depending on the route of administration.

[0067] The pharmaceutical compositions of the present invention are formulated to be compatible with their intended route of administration. For systemic use, the pharmaceutical compositions of the present invention can be formulated for administration by a conventional route selected from the group consisting of intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intracerebral, intracranial, intrapulmonary, intranasal, intraspinal, intrathecal, transdermal, transmucosal, oral, vaginal, and rectal.

[0068] In one embodiment, parenteral administration is the preferred route of administration. The pharmaceutical composition may be packaged in a glass or plastic ampoule, a disposable syringe, a sealed bag, or a multiple dose vial. In one embodiment, administration as an intravenous injection is the preferred route of administration. The formulation may be administered continuously by infusion or by bolus injection.

[0069] Pharmaceutical compositions of the present invention may be presented in unit dosage form, i.e., as physically discrete units intended as unitary dosages for the subjects to be treated.

[0070] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL, or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists.

[0071] The compositions of the present invention should be stable under the conditions of manufacture and storage. Furthermore, the compositions should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.

[0072] Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars (for example, mannitol, sorbitol, etc.), polyalcohols, or sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[0073] Sterile injectable solutions of the pharmaceutical compositions of the present invention can be prepared by incorporating the required amount of active molecule in a suitable solvent with one or a combination of the above-mentioned ingredients, followed by sterile filtration.For sterile powders for preparing sterile injectable solutions, methods include vacuum drying and freeze-drying, whereby powders containing active ingredient and any desired additional ingredients are obtained from the solution previously sterile-filtered.

[0074] Except insofar as any conventional media or agent is incompatible with the active molecules of the present invention, its use in the compositions is contemplated within the scope of the present invention.

[0075] In one embodiment, transferrin, lactoferrin, or a combination thereof may comprise at least 20% by weight of the total protein content of a pharmaceutical composition of the invention. For example, transferrin, lactoferrin, or a combination thereof may comprise about 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99% or more by weight of the total protein content of a pharmaceutical composition of the invention.

[0076] Those skilled in the art will understand that the specific embodiments disclosed in the above paragraphs should not be read in isolation, and that the specification intends for these embodiments to be disclosed in combination with other embodiments, not individually. For example, each of the embodiments disclosed in the above paragraphs should be read as expressly combined with each of the embodiments in the above paragraphs, or with two or more of the embodiments disclosed therein, in any order.

[0077] dosage As discussed above, the present inventors speculate that by increasing the concentration of transferrin, lactoferrin, or a combination thereof near the site of neurodegenerative injury or damage, such molecules can enhance and / or promote the body's own nerve regeneration and repair mechanisms. Transferrin and lactoferrin may be administered directly or indirectly to the site of neurodegenerative injury or damage by any conventional drug delivery means known to those skilled in the art. For example, transferrin, lactoferrin, or a combination thereof may be administered locally to or near the damage caused by a neurodegenerative event by a conventional route selected from the group consisting of intracerebral, intracranial, intraspinal, and intrathecal. For example, transferrin, lactoferrin, and a combination thereof may be administered locally during surgical intervention.

[0078] Alternatively, transferrin, lactoferrin, or a combination thereof could be delivered indirectly to the site of neurodegenerative injury or damage by a route of administration selected from the group consisting of intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intrapulmonary, intranasal, transdermal, transmucosal, oral, intravaginal, and intrarectal.

[0079] To avoid any misunderstanding, we would like to clarify here that we are referring to transferrin iron saturation levels in two separate and distinct contexts: a) In the first context, as outlined above, the present specification refers to the iron saturation of purified exogenous transferrin in a pharmaceutical composition to be administered to a patient. In this case, the iron saturation level of the purified exogenous transferrin can be determined using inductively coupled plasma atomic emission spectroscopy (although other methods, such as colorimetry, can also be used). b) In a second context, which will be discussed in more detail immediately below, the present specification describes measuring the iron saturation of physiological transferrin in a patient, i.e., in the patient's plasma or serum, after a pharmaceutical composition containing exogenous transferrin has been administered to the patient.

[0080] Under normal physiological conditions, substantially all iron in plasma is bound to transferrin, resulting in an iron saturation of physiological transferrin of approximately 30%. In Example 6 (see below), the inventors demonstrated that transferrin with an iron saturation of less than 30% can provide unexpected neuroregenerative effects. As a non-limiting hypothesis, it is hypothesized that administering a pharmaceutical composition containing exogenous transferrin (with a low iron saturation) to a patient increases the physiological concentration of transferrin in the patient's plasma, thereby reducing the iron saturation of physiological transferrin to less than 30%. Thus, physiological transferrin can be utilized to exert its neuroregenerative effects. Naturally, exogenous transferrin with an iron saturation of less than 1% is likely to be more effective than exogenous transferrin with an iron saturation of 40%.

[0081] Thus, in one embodiment, a protein selected from transferrin, lactoferrin, and a combination thereof is administered to a patient at a concentration sufficient to reduce the iron saturation of transferrin (in the patient's serum or plasma sample) to less than about 30%. Preferably, a protein selected from transferrin, lactoferrin, and a combination thereof is administered to a patient at a concentration sufficient to reduce the iron saturation of transferrin (in the patient's serum or plasma sample) to less than about 20%, for example, less than about 10%. Transferrin, lactoferrin, or a combination thereof may be administered to a patient using a dosage regimen based on dose adjustment to achieve this level of serum or plasma transferrin iron saturation.

[0082] Those skilled in the art will appreciate that measuring transferrin iron saturation levels in a patient's serum or plasma is a routine assay that is typically performed using colorimetric methodology as discussed above. The iron content of plasma or serum is measured in a chemical analyzer by using a colorimetric reaction that uses ferrene or ferrozine as a chromogen to form a colored complex with iron. The analyzed sample will produce two values: The iron content of the sample (i.e., the iron bound to transferrin in the sample) and the unsaturated iron binding capacity (UIBC, i.e., the number of unoccupied iron binding sites on transferrin in the sample). Total iron binding capacity (TIBC) is the sum of the iron content of a sample and the UIBC. Transferrin saturation (%) is determined as [(iron content of sample / TIBC) x 100].

[0083] The work of colorimetric assays for measuring transferrin iron saturation levels in patient serum or plasma is common knowledge, and further information can be found in various literature reviews, such as Pfeiffer et al., Am J Clin Nutr 2017, 106(Suppl), 1606S-14S, the contents of which are incorporated herein by reference.

[0084] In still further embodiments of the methods of the present invention, a protein selected from transferrin, lactoferrin, and combinations thereof can be administered to a patient at a concentration of about 5 mg / kg to about 8400 mg / kg. For example, about 10 mg / kg to about 7000 mg / kg, e.g., about 20 mg / kg to about 6000 mg / kg, e.g., about 50 mg / kg to about 5000 mg / kg. In some embodiments, a protein selected from transferrin, lactoferrin, and combinations thereof can be administered to a patient at a concentration of about 50 mg / kg to about 1000 mg / kg. Suitably, the protein can be administered at a concentration of about 50 mg / kg to about 500 mg / kg, e.g., about 50 mg / kg to about 250 mg / kg, e.g., about 50 mg / kg to about 150 mg / kg.

[0085] In one embodiment, the method of the present invention may include administering a protein selected from transferrin, lactoferrin, and combinations thereof to a patient in need thereof as part of a multiple-dose regimen. For example, about 50 mg / kg to about 5,000 mg / kg for the first dose on day 1 of the administration period, followed by about 50 mg / kg to about 1,000 mg / kg per dose during the multiple-dose period. For example, about 50 mg / kg to about 1,000 mg / kg for the first dose on day 1 of the administration period, followed by about 50 mg / kg to about 500 mg / kg per dose during the multiple-dose period. For example, about 50 mg / kg to about 500 mg / kg for the first dose on day 1 of the administration period, followed by about 50 mg / kg to about 250 mg / kg per dose during the multiple-dose period. For example, about 50 mg / kg to about 250 mg / kg for the first dose on day 1 of the administration period, followed by about 50 mg / kg to about 250 mg / kg per dose during the multiple-dose period. The period of multiple dosing may include about 3 to about 30 administrations up to a total cumulative dose. The period of multiple dosing may be about 1 to about 30 weeks. Multiple partial doses may be administered at intervals of about 1 day to about 30 days.

[0086] Those skilled in the art will understand that the specific embodiments disclosed in the above paragraphs should not be read in isolation, and that the specification intends for these embodiments to be disclosed in combination with other embodiments, not individually. For example, each of the embodiments disclosed in the above paragraphs should be read as expressly combined with each of the embodiments in the above paragraphs, or with two or more of the embodiments disclosed therein, in any order.

[0087] Additional features and advantages of the present invention will become more apparent in the accompanying drawings. [Brief explanation of the drawings]

[0088] [Figure 1-1] Figure 1A is a graph showing the induction of neurite outgrowth in SH-SY5Y cells in response to apo-transferrin, and Figure 1B is a graph showing the induction of proliferation in SH-SY5Y cells in response to apo-transferrin. [Figure 1-2] Figure 1C is an image showing an increase in β-III-tubulin protein concentration in SH-SY5Y cells in response to apo-transferrin, and Figure 1D is a graph showing an increase in β-III-tubulin protein concentration in SH-SY5Y cells in response to apo-transferrin. [Figure 2] 1 is a graph demonstrating that apo-transferrin induces primary human neural progenitor cells to become β-III-tubulin protein-positive neurons and GFAP protein-positive astrocyte cells. [Figure 3] Figure 3A is a graph plotting the effect of various concentrations of deferoxamine mesylate relative to apo-transferrin on neurite outgrowth in SH-SY5Y cells, and Figure 3B is a graph showing the efficacy of transferrin mutants with reduced iron-binding capacity in promoting neurite outgrowth in SH-SY5Y cells. [Figure 4] 1 is a graph plotting the effect of various different proteins on neurite outgrowth in SH-SY5Y cells. [Figure 5] 1 is a graph plotting the effect of IOX2, a prolyl hydroxylase inhibitor, on neurite outgrowth in SH-SY5Y cells. [Figure 6] 1 is a graph showing the role of iron saturation on the efficacy of transferrin in promoting neurite outgrowth in SH-SY5Y cells. [Figure 7] FIG. 1 is a graph plotting the effect of apo-transferrin in combination with other neurotrophic protein / peptide fragments on neurite outgrowth in SH-SY5Y cells. [Figure 8] 1 is a graph plotting the effect of apo-transferrin in combination with the small molecule Y-27632 on neurite outgrowth in SH-SY5Y cells. [Figure 9]FIG. 1 is a graph showing that apo-transferrin increases the amount of neurogenesis as measured by new neuroblasts (defined by BrdU+ / DCX+ cells) and newly formed mature neurons (defined by BrdU+ / NeuN+ cells) in an animal test model. [Figure 10-1] Figure 10A is a graph demonstrating that treating animals with apo-transferrin after transient MCAo results in faster recovery compared to saline-treated mice, and Figure 10B is a graph demonstrating that treating animals with apo-transferrin after transient MCAo results in better motor skills compared to saline-treated mice. [Figure 10-2] FIG. 10C is a graph demonstrating that treating animals with apo-transferrin after transient MCAo resulted in higher cognitive performance compared to saline-treated mice. DETAILED DESCRIPTION OF THE INVENTION

[0089] Detailed Examples of the Invention It should be readily apparent to one skilled in the art that the examples disclosed herein below represent generalized examples only, and that other configurations and ways in which the present invention can be reproduced are possible and encompassed by the present invention. [Example]

[0090] Apo-transferrin (ApoTf) induces differentiation and neurite outgrowth in a dose-responsive manner in SH-SY5Y cells Transferrin is utilized to deliver iron as a nutrient to cells in cell culture and in vivo. This is typically achieved through the binding of holo-transferrin (HoloTf) to its cognate receptor, CD71, transferrin receptor 1 (TfR1), and subsequent endocytosis. Transferrin is typically thought to provide iron to cells as a means of promoting and sustaining metabolic activity. The present inventors surprisingly found that apo-transferrin, a non-iron-containing form of the transferrin protein, induces differentiation of SH-SY5Y cells, a highly popular neuronal research model. Induction of neuronal differentiation was assessed by morphological parameters of neurite formation (key elements typically used as markers of neuronal differentiation, neuronal health, and function) according to the procedures of Agholme, 2010. J. of Alzheimer's Disease. Vol. 20:1 p069-108; and Dyberg et al., 2017. PNAS Vol. 114(32), E6603-E6612.

[0091] Undifferentiated SH-SY5Y cells were seeded in 96-well clear-bottom plates in medium containing 0.1% FBS. Serum-free basal medium was used as recommended by the SH-SY5Y cell supplier (Sigma, catalog number 94030304-1VL). 24 hours after cell seeding, a 3x stock solution of ApoTf (final concentration in serum-free basal medium indicated on the x-axis) was added to the cells. ApoTf was obtained and purified from pooled human plasma and dosed at a final concentration of 0.2 mg / mL. Cells were allowed to differentiate for 6 days. Neurite outgrowth was assessed by imaging and image analysis. At the time of analysis, 10x solutions of Tubulin Tracker (Molecular Probes, T34075) and Hoechst 33342 (Molecular Probes #H3570) nuclear stains were prepared.

[0092] Briefly, Tubulin Tracker dissolved in DMSO was diluted 1:1 with Pluronic F-127 and further diluted in HBSS to create a 10x solution. Hoechst 33342 was added to the HBSS-Tubulin Tracker solution at 10 μg / mL to create a 10x nuclear stain. This 10x staining solution (10 μL) was added directly to the treated assay wells and incubated at 37°C for 30 minutes. Following incubation, 110 μL of 0.4% trypan blue was added directly to the assay wells and imaged using a Molecular Devices Nano imager. Nine images per well were acquired in the blue (nuclear) and green (tubulin) fluorescent channels for each image.

[0093] After image acquisition, cells, somata, and neurites were identified and quantified using the MetaExpress Neurite Outgrowth Analysis Module (Molecular Devices). To account for the different numbers of cells in each test well, the total number of neurite branches was divided by the total number of cells imaged. Fold changes in outgrowth were determined by setting untreated control cells to a value of 1 and expressing all other treatments relative to the untreated control.

[0094] It is clear from Figure 1A that apo-transferrin was able to induce neurite outgrowth in a dose-dependent manner. Gradually increasing apo-transferrin up to 0.8 mg / mL enhanced the outgrowth response in SH-SY5Y cells. This phenomenon is counterintuitive to the known function of transferrin, which acts primarily in the holo- or iron-loaded form.

[0095] Figure 1B shows that apo-transferrin induces a concentration-dependent increase in cell number, indicating increased cell proliferation, up to the highest dose tested, 0.8 mg / mL apo-transferrin.

[0096] Figure 1C provides a visual comparison of SH-SY5Y cells treated with 0.1 mg / mL ApoTf (lower panel) versus untreated controls (upper panel). The left panel shows nuclear staining with Hoechst 33342. The right image shows tubulin staining of cell bodies and neurites. From Figure 1C, it is clear that ApoTf had a profound effect on promoting cell proliferation and the subsequent / concomitant induction of neurite / tubulin outgrowth.

[0097] Additionally, as shown in Figure 1D, we found that treatment with apo-transferrin caused an increase in the well-characterized β-III-tubulin protein, a conventional marker of neurons. In this experiment, SH-SY5Y cells were differentiated as described above. At the time of analysis, cells were fixed with paraformaldehyde, stained for β-III-tubulin (R&D Systems, MAB1195), and imaged using a Molecular Devices Nano imager. Image analysis was performed by assessing the fluorescence intensity of β-III-tubulin-stained cells. Background from the secondary antibody alone was subtracted from all values. Values ​​for the indicated conditions are shown as "β-III-tubulin staining intensity" along with standard deviations.

[0098] SH-SY5Y cells "SH-SY5Y cells" herein refers to a subcloned cell line derived from the SK-N-SH neuroblastoma cell line. These cells can be converted into various types of functional neurons by the addition of specific compounds, and therefore serve as a model for neurodegenerative disorders. Additionally, the SH-SY5Y cell line has been widely used in experimental neurological research, including the analysis of neuronal differentiation, metabolism, and function in relation to neurodegenerative processes, neurotoxicity, and neuroprotection.

[0099] Outlined hereinafter are peer-reviewed citations that refer to the SH-SY5Y cell line as a predictive model for various neurodegenerative disorders. This list does not constitute an admission by the inventors of the prior art, but rather serves to illustrate the knowledge of those skilled in the art of the SH-SY5Y cell line as a predictive model for brain injury and neurological disorders.

[0100] Neurogenesis Dayem et al. Biologically synthesized silver nanoparticles induce neuronal differentiation of SH-SY5Y cells via modulation of reactive oxygen species, phosphatases, and kinase signaling pathways. Biotechnol. J. 2014, 9, pp. 934-943. Fagerstrom et al. Protein Kinase C-epsilon Implicated in Neurite Outgrowth in Differentiating Human Neuroblastoma Cells. Cell Growth & Differentiation, Vol. 7, pp. 775-785, June 1996.

[0101] Peripheral nerve injury Han et al. Berberine. Promotes Axonal Regeneration in Injured Nerves of the Peripheral Nervous System. J Med Food 15 (4) 2012, pp. 413-417. Gold et al. Nonimmunosuppressant FKBP-12 Ligand Increases Nerve Regeneration. EXPERIMENTAL NEUROLOGY 147, pp. 269-278 (1997). Kim et al. Protective effect of GCSB-5, an herbal preparation, against peripheral nerve injury in rats. Journal of Ethnopharmacology 136 (2011) pp. 297-304. Lesma et al. Glycosaminoglycans in Nerve Injury: I. Low Doses of Glycosaminoglycans Promote Neurite Formation. Journal of Neuroscience Research. 1996 46(5):565-71.

[0102] Diabetic neuropathy Hattangady and Rajadhyaksha. A brief review of in vitro models of diabetic neuropathy. Int J Diabetes Dev Ctries. 2009 Oct-Dec;29(4):143-149. Vincent et al. Oxidative Stress and Programmed Cell Death in Diabetic Neuropathy. Ann. NY Acad. Sci. 959: 368-383 (2002). Shindo. Modulation of Basal Nitric Oxide-dependent Cyclic-GMP Production by Ambient Glucose, Myo-Inositol, and Protein Kinase C in SH-SY5Y Human Neuroblastoma Cells. J. Clin. Invest. Vol. 97, No. 3, February 1996, pp. 736-745. Li et al. C-peptide enhances insulin-mediated cell growth and protection against high glucose-induced apoptosis in SH-SY5Y cells. Diabetes Metab Res Rev 2003; 19: pp. 375-385.

[0103] Anticancer drug-induced neuropathy Rigolio et al. Resveratrol interference with the cell cycle protects human neuroblastoma SH-SY5Y cell from paclitaxel-induced apoptosis. Neurochemistry International 46 (2005) pp. 205-211. Donzelli et al. Neurotoxicity of platinum compounds: comparison of the effects of cisplatin and oxaliplatin on the human neuroblastoma cell line SH-SY5Y. Journal of Neuro-Oncology 67: pp. 65-73, 2004. Mannelli et al. Oxaliplatin-induced oxidative stress innervous system-derived cellular models: Could it correlate with in vivo neuropathy? Free Radical Biology and Medicine 61 (2013) pp. 143-150.

[0104] Organophosphate-induced neurological disorders (pesticides, chemical warfare compounds) Hong et al. Neurotoxicity induced in differentiated SK-N-SH-SY5Y human neuroblastoma cells by organophosphorus compounds. Toxicology and Applied Pharmacology 186 (2003) 110 - 118 pages. Ehrich et al. Interaction of organophosphorus compounds with muscarinic receptors in SH-SY5Y human neuroblastoma cells. Journal of Toxicology and Environmental Health 1994 43(1):51 - 63 pages.

[0105] Traumatic brain injury Triyoso and Good. Pulsatile shear stress leads to DNA fragmentation in human SH-SY5Y neuroblastoma cell line. Journal of Physiology (1999), 515.2, 355 - 365 pages. Song et al. Arctigenin Confers Neuroprotection Against Mechanical Trauma Injury in Human Neuroblastoma SH-SY5Y Cells by Regulating miRNA-16 and miRNA-199a Expression to Alleviate Inflammation. J Mol Neurosci (2016) 60:115 - 129 pages. Skotak et al. An in vitro injury model for SH-SY5Y neuroblastoma cells: Effect of strain and strain rate. Journal of Neuroscience Methods 205 (2012) 159 - 168 pages. Arun et al. Studies on blast traumatic brain injury using in-vitro model with shock tube. NeuroReport (2011) 22:379-384.

[0106] ischemia Miglio et al. Cabergoline protects SH-SY5Y neuronal cells in an in vitro model of ischemia. European Journal of Pharmacology 489 (2004) pp. 157-165. Duong et al. Multiple protective activities of neuroglobin in cultured neuronal cells exposed to hypoxia re-oxygenation injury. J. Neurochem. (2009) 108, pp. 1143-1154. Qiu et al. Enhancement of ischemia-induced tyrosine phosphorylation of Kv1.2 by vascular endothelial growth factor via activation of phosphatidylinositol 3-kinase. J. Neurochem. (2003) 10.104. [Example]

[0107] Effects of ApoTf on β-III-tubulin and GFAP protein levels in primary human neural progenitor cells The neurogenic effects of ApoTf also apply to primary human cortical neural progenitor cells, another established model of adult neurogenesis (see Azari and Reynolds, "In Vitro Models for Neurogenesis." Cold Spring Harbor Perspective Biol 2016, 8, a021279). As shown in Figures 2A and 2B, apo-transferrin dramatically increases the percentage of cells differentiated into neurons (% β-III-tubulin-positive cells, 2A) and astrocytes (% GFAP-positive cells, 2B) from cultures of primary human cortical neural progenitor cells compared to cells lacking apo-transferrin.

[0108] Neural progenitor cells maintained as neurospheres were obtained from Lonza (PT-2599). Cells were thawed from frozen vials of neurospheres and cultured in Human NeuroCult™ NS-A Complete Proliferation Medium (Stemcell Technologies) for two weeks. Neurospheres were dissociated into single cells and plated onto laminin-coated wells of assay plates. Neural progenitor cells were seeded in NeuroCult™ NS-A basal medium containing 1 / 10 the recommended growth supplements in the absence or presence of ApoTf (0.8 mg / mL) for 72 hours. At the time of analysis, cells were fixed with paraformaldehyde, stained for β-III-tubulin (R&D Systems, MAB1195) and GFAP (Invitrogen, PA3-16727), and imaged using a Molecular Devices Nano imager. Image analysis was performed by assessing the relative number of cells staining positive for β-III-tubulin or GFAP. Values ​​for the indicated conditions are shown as "% β-III-tubulin positive" cells (Figure 2A) or "% GFAP positive" cells (Figure 2B) along with standard deviations. [Example]

[0109] Iron chelation is not the only mode of action for ApoTf-mediated neurogenesis Deferoxamine mesylate (DFO) is a small-molecule iron chelator used in the clinical practice of iron overload. Like ApoTf, DFO has a high affinity binding constant for iron but only a single iron-binding site. The effect of DFO on neurite outgrowth was investigated. ApoTf was tested at concentrations near the bottom of its functional dose curve and compared to DFO for its ability to induce neurite outgrowth. ApoTf tested at 2.4 μM (0.2 mg / mL) has two iron-binding sites, equivalent to 4.8 μM DFO, which has a single iron-binding site.

[0110] Undifferentiated SH-SY5Y cells were seeded and treated as described in Example 1. Neurite outgrowth was assessed by imaging and image analysis. At the time of analysis, a 10x solution of Tubulin Tracker (Molecular Probes, T34075) and Hoechst 33342 (Molecular Probes #H3570) nuclear stain was prepared. Briefly, Tubulin Tracker dissolved in DMSO was diluted 1:1 with Pluronic F-127 and further diluted in HBSS to create a 10x solution. Hoechst 33342 was added to the HBSS-Tubulin Tracker solution at 10 μg / mL to create a 10x nuclear stain. This 10x staining solution (10 μL) was added directly to the treated assay wells and incubated at 37°C for 30 minutes. Following incubation, 110 μL of 0.4% trypan blue was added directly to the assay wells and imaged using a Molecular Devices Nano imager. Nine images per well were acquired in the blue (nuclei) and green (tubulin) fluorescent channels for each image. After image acquisition, cell bodies were identified and neurites were quantified using the MetaExpress Neurite Outgrowth Analysis Module (Molecular Devices). To account for different cell numbers, the total number of neurite branches was divided by the total number of cells imaged. Fold changes in outgrowth were determined by setting untreated control cells to a value of 1 and plotting all other treatments relative to the untreated control. ApoTf was obtained and purified from pooled human plasma and dosed at a final concentration of 0.2 mg / mL. Deferoxamine mesylate (DFO) was obtained from Tocris (catalog no. 5764) and resuspended and stored according to the manufacturer's recommendations. The concentrations of DFO assessed for neurogenic properties are indicated on the x-axis.

[0111] Figure 3 shows that DFO exhibits maximal neurite outgrowth between 1 and 3 μM, with little neurite formation above that concentration, whereas ApoTf continues to increase differentiation up to 9.9 μM (0.8 mg / mL; 20 μM iron-binding sites). These data suggest that while iron chelation may play a role in neurite outgrowth, it is not the primary mechanism of action and that other, as yet unidentified, functional aspects of ApoTf must also play a role in its neurogenic potential.

[0112] We further sought to determine whether reduced iron-binding activity of transferrin by mutation of the N-terminal iron-binding site was sufficient to mediate neurogenesis. Undifferentiated SH-SY5Y cells were treated as described in Example 1. Neurite outgrowth was assessed by imaging and image analysis. At the time of analysis, a 10x solution of Tubulin Tracker (Molecular Probes, T34075) and Hoechst 33342 (Molecular Probes #H3570) nuclear stain was prepared. Briefly, Tubulin Tracker dissolved in DMSO was diluted 1:1 with Pluronic F-127 and further diluted in HBSS to create a 10x solution. Hoechst 33342 was added to the HBSS-Tubulin Tracker solution at 10 μg / mL to create a 10x nuclear stain. This 10x staining solution (10 μL) was added directly to the treated assay wells and incubated at 37°C for 30 minutes. Following incubation, 110 μL of 0.4% trypan blue was added directly to the assay wells and imaged using a Molecular Devices Nano imager. Nine images per well were acquired in blue (nuclei) and green (tubulin) fluorescent channels for each image. After image acquisition, cell bodies were identified and neurites were quantified using the MetaExpress Neurite Outgrowth Analysis Module (Molecular Devices).

[0113] To account for different cell numbers, the total number of neurite branches was divided by the total number of cells imaged. The untreated control was set to a value of 1, and all other treatments were expressed relative to the untreated control to determine the fold change in outgrowth. All proteins were dosed at a final concentration of 0.2 mg / mL.

[0114] Plasma-derived human serum albumin (pdHSA) and ApoTf were obtained and purified from pooled human plasma. Recombinant ApoTf (rec ApoTf; SEQ ID NO: 1) and N-lobe mutant Tf (N-mut rec ApoTf; SEQ ID NO: 4) were obtained by cell culture expression from 293-6E cells.

[0115] Briefly, wild-type human transferrin (SEQ ID NO: 1) and N-lobe mutant human transferrin (SEQ ID NO: 4) sequences were cloned into mammalian expression plasmids containing an N-terminal 6xHIS tag and a TEV cleavage site. These expression plasmids were transfected into the 293-6E cell line, and proteins were then collected from the cell culture supernatant. The proteins were purified on an Ni-NTA column and eluted after washing. TurboTEV protease was used to cleave the N-terminal 6xHIS tag and additional amino acids from the transferrin protein. Following TEV cleavage, the transferrin protein was separated from the cleaved 6xHIS tag and uncleaved protein by a second Ni-NTA capture column. The flow-through fraction from the Ni-NTA capture column was then subjected to a low pH treatment to remove any remaining iron that may be bound to these proteins, buffer exchanged into PBS pH 7.4, concentrated, and sterile filtered for final use.

[0116] Figure 3B shows that plasma-derived human serum albumin (pdHSA) had no effect on neurogenesis. However, both ApoTf and recombinant ApoTf induced neurogenesis in SH-SY5Y cells. An ApoTf mutant with reduced iron-binding capacity (N-mut rec ApoTf) was nearly equivalent to ApoTf and rec ApoTf in inducing differentiation of SH-SY5Y cells. Iron binding alone does not appear to be the mechanism of action for the neurogenic potential of ApoTf. [Example]

[0117] Neurogenic effects on SH-SY5Y cells are specific to apo-transferrin and apo-lactoferrin Since the role of iron chelation in the neurogenic potential of ApoTf was unclear from Example 3, we determined whether other iron-binding proteins could also mediate neurogenesis in SH-SY5Y cells.

[0118] Undifferentiated SH-SY5Y cells were treated as described in Example 1. Neurite outgrowth was assessed by imaging and image analysis. At the time of analysis, a 10x solution of Tubulin Tracker (Molecular Probes, T34075) and Hoechst 33342 (Molecular Probes #H3570) nuclear stain was prepared. Briefly, Tubulin Tracker dissolved in DMSO was diluted 1:1 with Pluronic F-127 and further diluted in HBSS to create a 10x solution. Hoechst 33342 was added to the HBSS-Tubulin Tracker solution at 10 μg / mL to create a 10x nuclear stain. This 10x staining solution (10 μL) was added directly to the treated assay wells and incubated at 37°C for 30 minutes. Following incubation, 110 μL of 0.4% trypan blue was added directly to the assay wells and imaged using a Molecular Devices Nano imager. Nine images per well were acquired in the blue (nuclei) and green (tubulin) fluorescent channels for each image. After image acquisition, cell bodies were identified and neurites were quantified using the MetaExpress Neurite Outgrowth Analysis Module (Molecular Devices). To account for different cell numbers, the total number of neurite branches was divided by the total number of cells imaged. The untreated control was set to a value of 1, and all other treatments were expressed relative to the untreated control to determine fold changes in outgrowth. BSA was obtained from Sigma. rHSA was obtained from Albumedix. ApoTf and HoloTf were obtained and purified from pooled human plasma. Apo-ferritin (horse) was obtained from Sigma. Apo-lactoferrin was obtained from Athens Research & Technology. All proteins were dosed at a final concentration of 0.2 mg / mL.

[0119] Figure 4 shows that neither bovine serum albumin (BSA) nor the low-affinity iron-binding form of human serum albumin affected neurogenesis. For more information on the low-affinity iron-binding form of human serum albumin (rHSA), see Silva et al., 2009. Biochimica et Biophysica Acta, 1794, 1449-1458. Holo-transferrin (HoloTf), an iron-saturated form of transferrin, also failed to induce differentiation of SH-SY5Y cells.

[0120] Surprisingly, apo-ferritin, an iron-deficient form of ferritin, another high-affinity iron-binding protein with multiple iron-binding sites, was ineffective in inducing differentiation of SH-SY5Y cells. This advances the hypothesis that iron binding alone is not the mechanism of action for the neurogenic potential of ApoTf. Unexpectedly, apo-lactoferrin also induced differentiation of these cells. Apo-lactoferrin is a structural and functional homolog of apo-transferrin, but is found in breast milk but not plasma.

[0121] Apo-lactoferrin shares 61% identity with apo-transferrin, whereas apo-ferritin and human serum albumin (HSA) are structurally unrelated to either apo-transferrin or apo-lactoferrin. [Example]

[0122] ApoTf-induced differentiation of SH-SY5Y cells is not mediated by hypoxia-inducible factor 1α (HIF-1α) Both ApoTf and HoloTf have been reported to be able to induce the production of HIF-1α, leading to associated neuroprotective effects (Grifols Worldwide Operations Limited, US2016008437, the contents of which are incorporated herein by reference). While this is a beneficial attribute before neurons die, neuroprotection does not benefit patients once nerve cells have died. Neurogenesis, on the other hand, can regenerate new nerve cells and therefore benefit patients even after injury.

[0123] In substantiating the premise that ApoTf mediates neurogenesis outside the HIF pathway, we tested a well-known, highly specific inhibitor of prolyl hydroxylase (PHD2) in an SH-SY5Y cell differentiation assay. IOX2 (N-[[1,2-dihydro-4-hydroxy-2-oxo-1-(phenylmethyl)-3-quinolinyl]carbonyl]-glycine), a small molecule inhibitor of PHD2, is known to activate the HIF pathway through its action on PHD2. See Chowdhury et al., 2013. ACS Chem. Biol. 8, 1488. IOX2 inhibited PHD2 with an IC50 of 22 nM. 50 It has the ability to induce upregulation of HIF-1α at concentrations as low as 1 μM in undifferentiated SH-SY5Y cells (Ross, US2016008437, supra).

[0124] Undifferentiated SH-SY5Y cells were seeded and treated as described in Example 1. Neurite outgrowth was assessed by imaging and image analysis. At the time of analysis, a 10x solution of Tubulin Tracker (Molecular Probes, T34075) and Hoechst 33342 (Molecular Probes #H3570) nuclear stain was prepared. Briefly, Tubulin Tracker dissolved in DMSO was diluted 1:1 with Pluronic F-127 and further diluted in HBSS to create a 10x solution. Hoechst 33342 was added to the HBSS-Tubulin Tracker solution at 10 μg / mL to create a 10x nuclear stain. This 10x staining solution (10 μL) was added directly to the treated assay wells and incubated at 37°C for 30 minutes. Following incubation, 110 μL of 0.4% trypan blue was added directly to the assay wells and imaged using a Molecular Devices Nano imager. Nine images per well were acquired in the blue (nuclei) and green (tubulin) fluorescent channels for each image. After image acquisition, cell bodies were identified and neurites were quantified using the MetaExpress Neurite Outgrowth Analysis Module (Molecular Devices). To account for different cell numbers, the total number of neurite branches was divided by the total number of cells imaged. Fold changes in outgrowth were determined by setting the untreated control to a value of 1 and plotting all other treatments relative to the untreated control. ApoTf was obtained and purified from pooled human plasma and dosed at a final concentration of 0.2 mg / mL. IOX2 was obtained from Tocris (catalog no. 4451) and resuspended and stored according to the manufacturer's recommendations.

[0125] It is clear from Figure 5 that neither neurite outgrowth nor differentiation was observed in IOX2-treated cells. Even at a very high concentration of 4 μM IOX2 (4-fold higher than the concentration reported in US2016008437 to induce HIF-1α in SH-SY5Y cells and the IC50 value reported by Chowdhury for PHD2 protein), no effect was observed. 50(More than 180-fold higher than the concentration determined as . These data, combined with the lack of neurogenesis in HoloTf (Example 4), indicate that HIF-1α does not play a role in the differentiation of SH-SY5Y cells. [Example]

[0126] The role of iron saturation in transferrin efficacy. ApoTf with various purities and iron saturations, as outlined in Table 1, were evaluated for their neurogenic potential. Transferrin samples were prepared according to procedures / methodologies known to those skilled in the art, as detailed in L. von Bonsdorff et al., Transferrin, Chapter 21, Section 21.4, pp. 301-310, in Production of Plasma Proteins for Therapeutic Use, edited by J. Bertolini et al., Wiley, 2013 [Print ISBN: 9780470924310 | Online ISBN: 9781118356807], the contents of which are incorporated herein by reference.

[0127] Protein purity was determined by SDS-PAGE. Iron saturation levels were determined using ICP-AES according to the procedure outlined in Manley et al., J Biol Inorg Chem (2009) 14:61-74, the contents of which are incorporated herein by reference.

[0128] [Table 1]

[0129] Undifferentiated SH-SY5Y cells were treated as described in Example 1. Neurite outgrowth was assessed by imaging and image analysis. At the time of analysis, a 10x solution of Tubulin Tracker (Molecular Probes, T34075) and Hoechst 33342 (Molecular Probes #H3570) nuclear stain was prepared. Briefly, Tubulin Tracker dissolved in DMSO was diluted 1:1 with Pluronic F-127 and further diluted in HBSS to create a 10x solution. Hoechst 33342 was added to the HBSS-Tubulin Tracker solution at 10 μg / mL to create a 10x nuclear stain. This 10x staining solution (10 μL) was added directly to the treated assay wells and incubated at 37°C for 30 minutes. Following incubation, 110 μL of 0.4% trypan blue was added directly to the assay wells and imaged using a Molecular Devices Nano imager. Nine images per well were acquired in the blue (nuclei) and green (tubulin) fluorescent channels for each image. After image acquisition, the MetaExpress Neurite Outgrowth analysis module (Molecular Devices) was used to identify cell bodies and quantify neurites. To account for different cell numbers, the total number of neurite branches was divided by the total number of cells imaged. The untreated control was set to a value of 1, and all other treatments were expressed relative to the untreated control to determine fold changes in outgrowth.

[0130] Figure 6A plots the effect of ApoTf AD, with the purity and iron content outlined in Table 1, on neurite outgrowth in SH-SY5Y cells, dosed at a final concentration of 0.2 mg / mL. Figure 6B plots the effect of transferrin with various iron saturation levels (listed on the x-axis), dosed at a final concentration of 0.2 mg / mL, on neurite outgrowth in SH-SY5Y cells.

[0131] ApoTf (<0.3% saturation) and HoloTf (100% saturation) were prepared after purifying transferrin from pooled human plasma as outlined in von Bonsdorff (see above). Various iron saturation contents were generated by mixing ApoTf and HoloTf to generate the indicated percentage saturation plotted in Figure 6B.

[0132] Figure 6A shows that all ApoTf preparations (ApoTf A–D) were able to induce neurogenic differentiation of SH-SY5Y cells, even though the samples had a protein purity of only 94%. Figure 6B shows the extent to which iron saturation affects the ability of transferrin to induce differentiation of SH-SY5Y cells. In this example, ApoTf or HoloTf with a protein purity of at least 99% were mixed in various ratios to determine the effect of iron saturation / content. Transferrin with an iron saturation content of less than 30% exhibited neurogenic potential. [Example]

[0133] Apo-transferrin acts synergistically with neurotrophic proteins and peptide factors to induce differentiation Several neurotrophic protein factors are being considered for clinical use to stimulate neurogenesis in neurodegenerative conditions and after traumatic brain injury. See Houlton et al., 2019. Frontiers in Neurosci., Vol. 13, Paper 790; Weissmiller and Wu, 2012. Translational Neurodegeneration, Vol. 1:14; Apfel, 2001. Clin Chem Lab Med., Vol. 39(4), p. 351.

[0134] Proteins from three neurotrophic superfamilies were tested for function in combination with ApoTf. These neurotrophic proteins are BDNF (brain-derived neurotrophic factor; NGF superfamily), GNDF (glial cell line-derived neurotrophic factor; TGF-β superfamily), and CNTF (ciliary neurotrophic factor-1; neurokine superfamily). In addition, another known neurotrophic peptide, PACAP (amino acids 1–38 of pituitary adenylate cyclase-activating polypeptide), was evaluated for function in combination with ApoTf.

[0135] Undifferentiated SH-SY5Y cells were treated as described in Example 1. Neurite outgrowth was assessed by imaging and image analysis. At the time of analysis, a 10x solution of Tubulin Tracker (Molecular Probes, T34075) and Hoechst 33342 (Molecular Probes #H3570) nuclear stain was prepared. Briefly, Tubulin Tracker dissolved in DMSO was diluted 1:1 with Pluronic F-127 and further diluted in HBSS to create a 10x solution. Hoechst 33342 was added to the HBSS-Tubulin Tracker solution at 10 μg / mL to create a 10x nuclear stain. This 10x staining solution (10 μL) was added directly to the treated assay wells and incubated at 37°C for 30 minutes. Following incubation, 110 μL of 0.4% trypan blue was added directly to the assay wells and imaged using a Molecular Devices Nano imager. Nine images per well were acquired in the blue (nuclei) and green (tubulin) fluorescent channels for each image. After image acquisition, the MetaExpress Neurite Outgrowth analysis module (Molecular Devices) was used to identify cell bodies and quantify neurites. To account for different cell numbers, the total number of neurite branches was divided by the total number of cells imaged. The untreated control was set to a value of 1, and all other treatments were expressed relative to the untreated control to determine fold changes in outgrowth.

[0136] In Figures 7A-7D, ApoTf was dosed at a final concentration of 0.1 mg / mL, either alone or in combination with the indicated neurotrophic factors. (A) BDNF was obtained from Peprotech (catalog no. 450-02) and dosed at 25 ng / mL. (B) GDNF was obtained from Peprotech (catalog no. 450-10) and dosed at 1000 ng / mL. (C) CNTF was obtained from Peprotech (catalog no. 450-13) and dosed at 250 ng / mL. (D) PACAP was obtained from Tocris (catalog no. 1186) and dosed at 200 nM. The abbreviation SF stands for serum-free medium.

[0137] 7A-7D, it is clear that each of the neurotrophic factors and peptide fragments induced differentiation of SH-SY5Y cells to different degrees. In some cases, such as BDNF, differentiation was not induced by the neurotrophic factors at the concentrations tested in the absence of ApoTf. In all of the experiments presented, neurotrophic factors in combination with ApoTf induced more differentiation than the molecules tested alone. Unexpectedly, ApoTf exhibits synergistic effects with other neurotrophic factors and peptides on neurite outgrowth in SH-SY5Y cells. [Example]

[0138] Apo-transferrin acts synergistically with neurogenic small molecules to induce differentiation The ability of ApoTf to interact with non-protein-based neurogenic small molecule compounds was tested in Example 7. ApoTf was evaluated in combination with the neurogenic compound Y-27632 [trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride]. Y-27632 is a Rock 1 and Rock 2 (Rho kinase) inhibitor. Inhibition of Rock 1 and Rock 2 by small molecules has a known ability to induce neuronal differentiation, including in SH-SY5Y cells. See Dyberg et al., 2017, PNAS 114(32), E6603-E6612.

[0139] Undifferentiated SH-SY5Y cells were treated as described in Example 1. Neurite outgrowth was assessed by imaging and image analysis. At the time of analysis, a 10x solution of Tubulin Tracker (Molecular Probes, T34075) and Hoechst 33342 (Molecular Probes #H3570) nuclear stain was prepared. Briefly, Tubulin Tracker dissolved in DMSO was diluted 1:1 with Pluronic F-127 and further diluted in HBSS to create a 10x solution. Hoechst 33342 was added to the HBSS-Tubulin Tracker solution at 10 μg / mL to create a 10x nuclear stain. This 10x staining solution (10 μL) was added directly to the treated assay wells and incubated at 37°C for 30 minutes. Following incubation, 110 μL of 0.4% trypan blue was added directly to the assay wells and imaged using a Molecular Devices Nano imager. Nine images per well were acquired in the blue (nuclei) and green (tubulin) fluorescent channels for each image. After image acquisition, cell bodies were identified and neurites were quantified using the MetaExpress Neurite Outgrowth Analysis Module (Molecular Devices). To account for different cell numbers, the total number of neurite branches was divided by the total number of cells imaged. Fold changes in outgrowth were determined by setting the untreated control to a value of 1 and expressing all other treatments relative to the untreated control. ApoTf was dosed at a final concentration of 0.1 mg / mL, alone or in combination with the indicated small molecules. Y-27632 was obtained from Tocris (catalog no. 1254) and dosed at 50 μM.

[0140] Figure 8 shows that while Y-27632 itself is a potent neurogenic compound, in the presence of ApoTf the neurogenic effects were synergistic, exceeding the effects of either molecule alone. The ability of ApoTf to act synergistically with several known protein, peptide, and small molecule neurogenic entities is an unexpected, surprising, and intriguing finding. [Example]

[0141] Apo-transferrin promotes the formation of new neuroblasts and mature neurons in the brains of animals with transient MCAo C57BL / 6J mice (approximately 20 g) were anesthetized under isoflurane. After incision, a 6.0 silicone-coated monofilament suture was inserted into the external carotid artery to occlude the middle cerebral artery (MCAo). The occlusion was performed for 60 minutes under temperature control. Within 2 hours after the occlusion was released, animals were assessed on a 7-point "neuroscore" scale to identify visual indications of stroke. The scale ranges from 0 (no observable deficits) to 6 (moribund), taking into account the extension of the contralateral forelimb, severity of circling, loss of ambulation, and loss of consciousness. 0 = no observable defects 1 = Poor extension of the contralateral forelimb 2 = Mild turning behavior when tail is lifted, <50% tendency to turn to the contralateral side 3 = Mild, unchanged turning behavior, >50% attempt to turn to the contralateral side 4 = Consistent strong turning, mouse remains in the turning position for more than 1-2 seconds, its nose nearly reaching its tail 5 = Severe rolling with fall toward the side contralateral to the infarct, loss of walking or righting reflex, and 6 = Decreased level of consciousness, coma, or near death

[0142] Only animals with a "neurological score" of 4 or higher were included in further studies (n = 8-10 animals / group). Six hours after occlusion, mice were injected daily with 350 mg / kg apo-transferrin (intraperitoneal injection) or an equal volume of saline, along with 50 mg / kg bromodeoxyuridine (BrdU; Sigma-Aldrich Chemical Co., St. Louis, MO), for a total of 7 days, both delivered intraperitoneally. Body weight changes were monitored daily.

[0143] At the time points indicated in Example 9, brains were prepared for analysis by transcardial perfusion with ice-cold heparinized saline (2.5 IU / ml heparin) to remove blood from the brain. Fresh brains were removed, leaving the right cerebellum attached to the entire left hemisphere to aid in positioning the brain block during sectioning. The entire left hemisphere block was placed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at +4°C for 24 hours and then cryoprotected in 30% sucrose in 0.1 M PB for 2-3 days at +4°C on a shaker. The brain was then blotted to remove excess liquid, placed on the cork of a vial, and frozen in liquid nitrogen. The block was then stored at -80°C until cryostat sections were obtained. Neurogenesis was assessed by immunohistochemistry using antibodies against BrdU and doublecortin (DCX) to quantify new neuroblasts, or antibodies against BrdU and NeuN to quantify new neurons in the dentate gyrus of the brain.

[0144] Figure 9A demonstrates that ApoTf administration increases the number of neuroblasts over a two-week period, while Figure 9B shows that after four weeks, ApoTf-treated mice have a higher number of newly formed mature neurons. These data suggest that neuroblasts generated early in neurogenesis can continue to differentiate to give rise to new mature neurons. Thus, these results suggest that apo-transferrin can promote aspects of neurogenesis beyond those normally induced in response to ischemic stroke. [Example]

[0145] Apo-transferrin promotes recovery, motor skills, and cognition in a mouse model of transient MCAo stroke Mice were prepared as described in Example 9 above and evaluated as indicated at 3 days and 1, 2, 3, and 4 weeks after MCAo (n = 8-10 animals / group). Motor coordination (i.e., balance behavior and locomotor ability depending on the function of the corticostriatal system) was assessed using the rotarod test. Learning and memory ability was measured using the NORT (Novel Object Recognition Test; e.g., Anglada-Huguet et al., 2014, Molecular Neurobiology, Vol. 49, pp. 784-795; Denninger et al., 2018, J. Vis. Exp., Vol. 141, e58593).

[0146] Figure 10A shows that ApoTf significantly improves the rate at which animals recover after MCA occlusion (MCAo) over the course of 3 to 14 days, and possibly longer. As described in Example 9, all animals in this study had an initial neurological score of 4 or greater when assessed 2 hours after MCAo. The percentage of animals without observable deficits (i.e., a neurological score of "0" as described in Example 9) is shown as a function of time after MCAo, shown on the x-axis.

[0147] Figure 10B shows that animals treated with apo-transferrin after MCAo have increased motor and balance skills. The time it takes for animals to fall off the rotarod (latency to fall) is plotted against the time after MCAo. The mice's motor skills mirror those of the neurological scores in Figure 10A, with an increased rate of improvement in motor / balance skills as measured by the time the animals remained on the rotarod apparatus. Although recovery, as measured by neurological scores, was similar in both groups by 4 weeks, ApoTf-treated mice had an overall better ability to remain on the rotarod. Figure 10C provides evidence that ApoTf administration increases learning and cognitive function for at least 2 weeks after protein administration. Discrimination (%) represents the animal's memory and is measured by the percentage of time the animal spent investigating a newly presented object compared to the time it spent investigating a previously presented object. Animals with better cognition and memory spent more time on the novel object due to their memory of the previously presented object. This is expressed as increased % discrimination. ApoTf-treated animals have a higher "% discrimination" compared to saline-treated mice, suggesting that apoTf-treated mice recover their cognitive abilities better after MCAo.

[0148] Taken together, the data in Figures 10A-C suggest that apoTf-promoted neurogenesis leads to better motor and cognitive performance in subjects.

[0149] array The sequences mentioned above are outlined below in fasta format.

[0150] SEQ ID NO: 1: Human transferrin [UniProt Q06AH7] protein sequence

[0151] [ka]

[0152] SEQ ID NO: 2: Human lactoferrin [UniProt P02788] protein sequence

[0153] [ka]

[0154] SEQ ID NO: 3: Y188F transferrin N-lobe mutant protein

[0155] [ka]

[0156] SEQ ID NO: 4: Y95F / Y188F transferrin N-lobe mutant protein

[0157] [ka]

[0158] SEQ ID NO: 5: Y426F / Y517F transferrin C-lobe mutant protein

[0159] [ka]

[0160] SEQ ID NO: 6: BDNF

[0161] [ka]

[0162] SEQ ID NO: 7: GDNF

[0163] [ka]

[0164] SEQ ID NO: 8: CNTF

[0165] [ka]

[0166] SEQ ID NO: 9: PACAP

[0167] [ka]

Claims

1. A composition for use in a method for stimulating the development of new nerve cells by neurogenesis in patients suffering from neurodegenerative events resulting from at least one of traumatic brain injury, non-traumatic brain injury, spinal cord injury, peripheral nerve injury, or peripheral nerve disorder, The composition comprises a therapeutically effective amount of transferrin, wherein the transferrin has an iron saturation of less than 20%.

2. The composition according to claim 1, wherein the transferrin is human transferrin.

3. The composition according to claim 1 or 2, wherein the transferrin is derived from plasma or recombinant.

4. Recombinant transferrin, i) Y188F mutant containing the amino acid sequence described in Sequence ID No. 3; ii) Y95F / Y188F mutants containing the amino acid sequence described in SEQ ID NO: 4; iii) Y426F / Y517F mutants containing the amino acid sequence described in Sequence ID No. 5; and iv) These combinations The composition according to claim 3, wherein the mutant transferrin is selected from the group consisting of the following.

5. The composition according to any one of claims 1 to 4, wherein transferrin is the domain of the fusion protein and the fusion partner is an immunoglobulin Fc domain.

6. The composition according to any one of claims 1 to 5, wherein the traumatic brain injury or spinal cord injury is caused by at least one of a road traffic accident, assault, collision in sports, or an unprotected fall.

7. The composition according to any one of claims 1 to 6, wherein the non-traumatic brain injury is caused by at least one of ischemic stroke, hemorrhagic stroke, hypoxic encephalopathy, anoxic encephalopathy, ingestion of chemical toxins, hydrocephalus, meningitis, or encephalitis.

8. The composition according to any one of claims 1 to 7, wherein the neurodegenerative event results from a stroke selected from the group consisting of ischemic stroke and hemorrhagic stroke.

9. The composition according to any one of claims 1 to 8, administered in combination with a serum or plasma protein selected from the group consisting of albumin, alpha-1 antitrypsin / alpha-1 proteinase inhibitor, antithrombin, polyclonal immunoglobulin, polyspecific immunoglobulin, C1 esterase inhibitor, transthyretin, and combinations thereof.

10. The composition according to claim 9, wherein serum or plasma protein and transferrin are administered as unital dosage forms.

11. The composition according to any one of claims 1 to 10, administered in combination with a therapeutically effective amount of a neurogenesis or neurotrophic compound or molecule.

12. The composition according to claim 11, wherein the neurogenesis or neurotrophic compound or molecule is selected from the group consisting of members of the NGF superfamily, members of the TGF-β superfamily, members of the neurokine superfamily, neurotrophic peptides, Rho kinase inhibitors, and combinations thereof.

13. The composition according to claim 11 or 12, wherein a neurogenesis or neurotrophic compound or molecule and transferrin are administered as unital dosage forms.

14. Neurogenesis or neurotrophic compounds or molecules, Brain-derived neurotrophic factor containing the amino acid sequence described in Sequence ID No. 6, Glial cell line-derived neurotrophic factor containing the amino acid sequence described in Sequence ID No. 7, Ciliary neurotrophic factor-1 containing the amino acid sequence described in Sequence ID No. 8, PACAP containing the amino acid sequence described in Sequence ID No. 9, trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide and pharmaceutically acceptable salts thereof, Hexahydro-1-(5-isoquinolinyl-sulfonyl)-1H-1,4-diazepine and pharmaceutically acceptable salts thereof, as well as their combinations A composition according to any one of claims 11 to 13, selected from the group consisting of the following.

15. The composition according to any one of claims 1 to 14, wherein transferrin is administered to a patient in need via a route of administration selected from the group consisting of intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intracerebral, intracranial, intrapulmonary, intranasal, intraspinal, intramedullary, percutaneous, transmucosal, oral, vaginal, and rectal.

16. The composition according to any one of claims 1 to 15, wherein transferrin is administered locally or proximal to damage caused by a neurodegenerative event.

17. The composition according to any one of claims 1 to 16, wherein transferrin is administered to the patient at a concentration sufficient to reduce the patient's transferrin iron saturation to less than 30%.

18. The composition according to claim 17, wherein the iron saturation of the patient's transferrin is measured in a sample of the patient's serum or plasma.

19. The composition according to any one of claims 1 to 18, wherein transferrin is administered to the patient at a concentration of 5 mg / kg to 8400 mg / kg.

20. The composition according to any one of claims 1 to 19, wherein transferrin is administered to the patient as part of a multi-dose regimen.

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