Compositions for nerve regeneration

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 the use thereof in the field of regenerative medicine. In particular, disclosed herein are the application of transferrin and lactoferrin, and the use thereof in promoting the proliferation, induction, and / or differentiation of neural progenitor cells or neural stem cells in patients suffering from nerve injury.

Background Art

[0002] Injury to the brain and spinal cord can manifest as immediate or chronic neurodegenerative effects that drastically alter the quality of life of affected individuals. Acquired brain injury (ABI), i.e., injury not induced by heredity, congenital conditions, or birth, causes changes in neural activity in the brain that affect the physical integrity, metabolic activity, or functional capacity of nerve cells in the brain. Acquired brain injury exists in two types: 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 injury can be defined as non-penetrating or penetrating, and includes falls, assaults, motor vehicle accidents, and sports injuries. Non-traumatic brain injury (NBTI) causes damage to the brain through non-impact processes such as oxygen deprivation, exposure to toxins, and pressure from tumors. Examples of NTBI include lack of oxygen supply to the brain caused by stroke, aneurysm, and heart attack.

[0004] Stroke is one of the most common categories of NBTI, and occurs when brain tissue cannot obtain oxygen and nutrients due to interruption or reduction of blood supply to a part of the brain. The neurodegenerative process begins almost immediately, and brain cells start to die within minutes. Ischemic stroke occurs when a blood vessel (artery) supplying blood to a region of the brain becomes occluded by a thrombus. 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 is placing an enormous burden on healthcare infrastructure and costs. Currently, recombinant tissue plasminogen activator (rtPA) is the only treatment approved by the FDA for ischemic stroke. The main function of rtPA is to dissolve blood clots and promote reperfusion. The alternative method for re-establishing obstructed blood flow is surgical intervention.

[0006] rtPA treatment has a narrow treatment time window, and therefore its broad application is limited. Furthermore, while it is important to restore perfusion to ischemic tissue with rtPA, the cascade of necrosis, apoptosis, and inflammation begins within minutes of severe oxygen deprivation. Genetically programmed nerves (which can last from days to weeks) during inflammation of post-ischemic tissue. system There is a growing suspicion that cell death significantly contributes to the final pathology resulting from delays in patient treatment / evaluation. Therefore, even before patients participate in evaluation, permanent nerves... system This can also lead to damage to nerve cells.

[0007] As of this writing, there are no approved nerve regeneration therapies that can reverse the effects of neurodegeneration associated with TBI and NTBI. Given the lack of curative therapies, the majority of the literature in this field focuses on neuroprotection (administering specific molecules pre- or concurrently in response to anticipated neurodegenerative ischemia or reperfusion events). system It is not surprising that there is a bias towards non-recoverable methods (to mitigate cell death).

[0008] One such example is U.S. Patent Publication 2016008437, published by Grifols Worldwide Operations Ltd, which discloses apo-transferrin as exerting neuroprotective effects by modulating hypoxia-inducible factor (HIF) activity in a rat model of stroke. The inventors observed the neuroprotective effect of apo-transferrin, which manifested as a reduction in the volume of infarcted area in rats treated with apo-transferrin compared to control rats. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] U.S. Patent Publication No. 2016008437 [Non-patent literature]

[0010] [Non-Patent Document 1] Ceriotti et al., Improved direct specific determination of serum iron and total iron-binding capacity Clin Chem. 1980, 26(2), pp. 327-31. [Non-Patent Document 2] 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 [Non-Patent Document 3] 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] [Non-Patent Document 4] Arvidsson et al., 2002, Nat. Med., 8, pp. 963-970. [Non-Patent Document 5] Kokaia and Lindvall, 2003, Curr. Opin. Neurobiol., 13, pp. 127-132. [Non-Patent Document 6] Kernie et al., 2010, Neurobiol. Disease, 37, pp. 267-274. [Non-Patent Document 7] Remington: The Science and Practice of Pharmacy, 21st edition, 2005, edited by DB 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 (Appendix), 1606S~14S [Non-Patent Document 10] Agholme, 2010. Journal of Alzheimer's Disease. Vol. 20: 1, pp. 69-108; [Non-Patent Document 11] Dyberg et al., 2017. PNAS Vol. 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): pp. 565-571. [Non-Patent Document 18] Hattangady and Rajadhyaksha. A brief review of in vitro models of diabetic neuropathy. Int J Diabetes Dev Ctries. October-December 2009; 29(4): pp. 143-149. [Non-Patent Document 19] Vincent et al. Oxidative Stress and Programmed Cell Death in Diabetic Neuropathy. Ann. N.Y. Acad. Sci. 959: pp. 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. Volume 97, Issue 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 Literature 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: 65-73, 2004. [Non-Patent Literature 24] Mannelli et al. Oxaliplatin-induced oxidative stress in nervous system-derived cellular models: Could it correlate with in vivo neuropathy? Free Radical Biology and Medicine 61 (2013), pp. 143-150. [Non-Patent Literature 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 Literature 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., Vol. 13, Article 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. [Overview of the project] [Problems that the invention aims to solve]

[0011] Despite the above, it is clear that there are insufficient clinical candidates with the potential to reverse the debilitating effects of neurodegeneration associated with spinal cord injury, TBI, and NBTI. Focusing on this need, therapies that at least partially reverse traumatic and non-traumatic neurological injury have yet to be realized and are therefore highly desirable. [Means for solving the problem]

[0012] The words “comprise” and “having” are used herein in connection with the present invention to specify the presence of a described feature, complete, process, or component, but do not preclude the presence or addition of one or more other features, complete, process, component, or group thereof.

[0013] Those skilled in the art will understand that no particular embodiment disclosed herein should be read in isolation, and that this specification is intended to be read in combination with other embodiments, rather than individually. Accordingly, each embodiment may serve as a basis for modifying or limiting other embodiments disclosed herein.

[0014] Concentration, quantity, and other numerical data may be expressed or presented in the form of ranges in this specification. Such range forms are used for convenience and conciseness only, and should therefore be interpreted flexibly to include not only the numerical limits explicitly stated as the range limits, but also all individual numerical values ​​or subranges contained within that range, as if each numerical value and subrange were explicitly stated. For example, a numerical range of "10 to 100" should be interpreted to include not only the explicitly stated values ​​of 10 to 100, but also the individual values ​​and subranges within the range. Therefore, this numerical range includes individual values ​​such as 10, 11, 12, 13...97, 98, 99, 100, as well as subranges such as 10 to 40, 25 to 40, and 50 to 60. This same principle applies to ranges that state only one numerical value, such as "at least 10." Furthermore, such interpretation shall apply regardless of the width of the range or the characteristics described.

[0015] Treatment method In a first aspect, the present invention relates to a new neurological event 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. system A method for promoting and / or inducing cell generation, The present invention provides a method comprising the step of administering a therapeutically effective amount of protein, selected from transferrin, lactoferrin, and combinations thereof, to a patient who requires it.

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

[0017] Those skilled in the art will understand that, among the vast number of mammalian iron-binding proteins, transferrin and lactoferrin are related proteins of the transferrin family, sharing 61% sequence identity. In addition to several overlapping and complementary functions, transferrin and lactoferrin also exhibit several mutually exclusive functions. The present invention includes all wild-type mammalian transferrin proteins within its scope, but human transferrin (UniProtKB Sequence ID Q06AH7) containing the amino acid sequence described in Sequence ID 1 is particularly preferred. Similarly, the present invention includes all wild-type mammalian lactoferrin proteins within its scope, but human lactoferrin (UniProtKB Sequence ID P02788) containing the amino acid sequence described in Sequence ID 2 is particularly preferred.

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

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

[0020] "Transferrin" in this specification refers to a therapeutically effective dose of: • Wild-type (mammalian, preferably human) transferrin protein, • Its functional variant, • Its functional fragment, or • Those combinations This is interpreted as meaning...

[0021] The iron saturation of transferrin, 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, for example, about 10% or less. In some embodiments, the iron saturation is about 5% or less. In further embodiments, the iron saturation may be less than about 1%. To avoid any misunderstanding, the range presented herein as less than X% includes 0 to X%, i.e., transferrin with no iron bound at all, and an iron saturation of 0%.

[0022] As used herein, “apo-transferrin” means transferrin having an iron saturation of less than 1%. Similarly, “holo-transferrin” means transferrin having an iron saturation of 99% or more.

[0023] Those skilled in the art will understand that the iron saturation level of transferrin can be easily determined without undue burden by quantifying the total iron level in a sample with 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] Appropriate examples include the following: • Colorimetric assay - Iron is measured using Fe in ferrosin and acetate buffer. 2+ The intensity of the purple complex formed in the reaction between thiourea and Cu is quantified by measuring it at 562 nm. 2+It may be added to form complexes with contaminating metals such as ferrosin, which can also bind to ferrosin, potentially leading to 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), pp. 327-31 (its contents are incorporated herein by reference). • Inductively coupled plasma atomic emission spectroscopy (ICP-AES) is 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 cations and free electrons) to excite metal atoms / ions in a sample and analyzing the emission wavelength of electromagnetic radiation typical for that particular metal. While this technique is a standard analytical method within the realm of common technical knowledge for those skilled in the art, further 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 (its contents are incorporated herein by reference).

[0025] A preferred method for determining the iron content of a sample for the therapeutic method of the present invention is ICP-AES. Then, the iron saturation of transferrin is 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. Since wild-type human transferrin (molecular weight 79,750) can bind to two iron atoms, a sample containing 1 g of transferrin will be 100% saturated with 1.4 mg of iron.

[0026] If the transferrin concentration of a particular sample is unknown, it can be easily determined by various well-characterized immunological (ELISA, turbidimetry) and non-immunological methods (absorbance, AU480 chemical assay).

[0027] As of the time of writing, transferrin is not approved as a medicine in any major jurisdiction worldwide. Therefore, there is no pharmacopoeia monograph for transferrin. Further information on the physical properties of transferrin, such as iron saturation, can be obtained from the main reference books consulted by those skilled in the art. See L von Bonsdorff et al., Transferrin, Chapter 21, pp. 301-310, and Production of Plasma Proteins for Therapeutic Use, edited by J. Bertolini et al., Wiley, 2013 [Print ISBN: 9780470924310 Online ISBN: 9781118356807]. Its contents are incorporated herein by reference and are considered to be within the realm of common technical knowledge for those skilled in the art.

[0028] "Lactoferrin" in this specification refers to a therapeutically effective amount: • Wild-type (mammalian, preferably human) lactoferrin protein, • Its functional variant, • Its functional fragment, or • Those combinations This is interpreted as meaning...

[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, for example, about 10% or less. In some embodiments, the iron saturation is about 5% or less. In further embodiments, the iron saturation may be less than about 1%.

[0030] As used herein, "apo-lactoferrin" means lactoferrin with an iron saturation of less than 1%. Similarly, "holo-lactoferrin" means lactoferrin with an iron saturation of 99% or more. The iron content and saturation levels of lactoferrin can be measured in the same way as those of transferrin, which are discussed in detail above.

[0031] In using the terms transferrin and lactoferrin, this specification includes recombinant derivatives of transferrin and lactoferrin, which 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, deletions, or insertions that do not substantially alter the structure or hydrophobic / hydrophilic properties of the recombinant protein compared to the wild-type protein. Recombinant variants of transferrin and lactoferrin within the scope of the present invention may also include at least one post-translational modification, such as PEGylation, glycosylation, polysialylation, or a combination thereof.

[0032] In one embodiment, the present invention envisions recombinant variants of transferrin and lactoferrin having one or more conservative substitutions compared to the wild-type proteins in SEQ ID NOs: 1 and 2. A “conservative substitution” is defined as the substitution of one amino acid with another amino acid having similar properties, such that those skilled in peptide chemistry would predict that the secondary structure and hydrophobicity / hydrophilicity indicators of the polypeptide would not be substantially altered. Generally, changes among 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 method of the present invention may have at least 90%, 95%, 96%, 97%, 98%, or 99% homology to wild-type human transferrin and human lactoferrin protein as briefly described in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0034] In further embodiments, the present invention includes specific mutant transferrins and / or lactoferrins that maintain their structure but do not bind iron to iron via an iron-binding domain, such as an N-lobe, a C-lobe, or a combination thereof.

[0035] Transferrin variants within the scope of this invention include, but are not limited to, the following: 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 understand that recombinant proteins can be obtained using standard techniques well known in the field 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 selected host cells, 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 into the viral genome. Some vectors are capable of autonomous replication within the host cell into which they are introduced (e.g., vectors with replication origins that function within the host cell). Other vectors can be integrated into the host cell's genome when introduced into the host cell, thereby replicating with the host genome. Furthermore, some preferred vectors are capable of inducing the expression of genes into 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 Kuyveromyces spp. Suitable insect cells include those derived from silkworms (Bombyx mori), armyworms (Mamestra brassicae), Spodoptera frugiperda, nettle moths (Trichoplusia ni), and fruit flies (Drosophila melanogaster). Examples of 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, mouse L-929 cells, HaK hamster cell lines, mouse 3T3 cells derived from Swiss, Balb-c, or NIH mice, and CV-1 cell lines.

[0039] The present invention also intends to utilize 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 substance. For example, suitable fusion or conjugation partners include serum albumin (e.g., bovine, rabbit, or human), keyhole limpet hemocyanin, immunoglobulin molecules (containing 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 present invention, the transferrin and lactoferrin proteins used in the method of the present invention are fusion proteins having an improved in-vivo half-life, in this case, • A wild-type (mammalian, preferably human) transferrin or lactoferrin protein is fused to a fusion partner selected from an immunoglobulin Fc domain and albumin; or • A mutant transferrin or lactoferrin protein within the scope 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 include at least a portion of a steady-state heavy-chain immunoglobulin domain. The steady-state heavy-chain immunoglobulin domain is preferably an Fc fragment including CH2 and CH3 domains and optionally at least a portion of a 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 includes at least a portion of a steady-state IgG immunoglobulin Fc domain. The IgG immunoglobulin Fc domain may be selected from IgG1, IgG2, IgG3, or IgG4 Fc domains, or modified Fc domains thereof.

[0042] In one embodiment, the fusion protein may include transferrin fused to the IgG1 Fc domain. In another embodiment, the fusion protein may include a transferrin variant fused to the IgG1 Fc domain.

[0043] Neurodegenerative events Surprisingly, the inventors have found that transferrin and lactoferrin can transfer nerve cells from neural progenitor cells and / or neural stem cells. system We discovered that both proteins have an unexpected therapeutic role beyond iron binding / iron delivery to cells, as they were extremely effective in stimulating cell development. Therefore, the present invention relates to the neurological role of both proteins 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. system A method for stimulating cell development, The present invention provides a method comprising the step of administering a therapeutically effective amount of protein, selected from transferrin, lactoferrin, and combinations thereof, to a patient who requires it.

[0044] When used herein, "nerve" systemThe term "stimulating cell development" means that transferrin or lactoferrin stimulates the development of new nerve cells. system It is used to mean directly or indirectly affecting neural progenitor cells and / or neural stem cells in a patient to produce cells. Without intending to limit the generality of the present invention, administration of transferrin or lactoferrin, compared to neural progenitor cells / neural stem cells not exposed to transferrin or lactoferrin, i) Proliferation of neural progenitor cells and / or neural stem cells within the patient, ii) Induce differentiation of neural progenitor cells and / or neural stem cells, thereby differentiating the neural cells system Turning into cells It is presumed that at least one of these will consequently increase.

[0045] "nerves" system In this specification, "cells" include, but are not limited to, glial cells and all cells of the nervous system, including nerve cells. In one embodiment, the nerves referred to in the method of the present invention system The cells in question are nerve cells, and transferrin and lactoferrin enhance the formation of new nerve cells.

[0046] As used herein, the term "neurodegenerative event" also refers to a neurological event. system This causes loss of cellular structure and / or function, and nerve system This refers to an event that includes cell death. This event is an immediate nerve death. system It may be an isolated, one-off event / occurrence that causes cell damage or death. Alternatively, the event may be a nerve system These can be continuous or chronic events that gradually lead to an increasing level of cellular damage or death. In certain embodiments, neurodegenerative events cause loss of structure, loss of function, or death of nerve cells (or neurons) in the brain and / or spinal cord, resulting in damage and dysfunction in the brain and / or spinal cord.

[0047] With respect to the method of the present invention, the neurodegenerative event is the result of at least one of the following: 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 brain injury caused by penetrating or non-penetrating trauma to the head. There are many possible causes, including, but are not limited to, road traffic accidents, assaults, sports collisions, and unprotected falls.

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

[0050] Similarly, “spinal cord injury” is interpreted herein to mean damage to any part of the spinal cord or to the 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 have suffered a neurodegenerative event resulting from at least one of the following: stroke, peripheral nerve injury, traumatic brain injury, or peripheral neuropathy. For example, the patient may have suffered a neurodegenerative event resulting from peripheral nerve injury. In one embodiment, the patient may have suffered a neurodegenerative event resulting from a stroke, for example, ischemic stroke or hemorrhagic stroke. In one embodiment, the patient may have suffered a neurodegenerative event resulting from ischemic stroke.

[0052] As a non-restrictive / unrestrictive theory, it is known that neurodegenerative injury or damage migrates neural stem cells to the site of such injury or damage. See Arvidsson et al., 2002, Nat. Med., 8, pp. 963-970; Kokaia and Lindvall, 2003, Curr. Opin. Neurobiol., 13, pp. 127-132; and Kernie et al., 2010, Neurobiol. Disease, 37, pp. 267-274. The inventors hypothesize that by increasing the concentration of transferrin, lactoferrin, or a combination thereof in a patient, such molecules can 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 preceding paragraphs should not be read in isolation, and that this specification is intended to disclose these embodiments not individually, but in combination with other embodiments. For example, each of the embodiments disclosed in the preceding paragraphs should be read as being expressly combined with each of the embodiments in the preceding paragraphs, or with any two or more embodiments disclosed therein in any order.

[0054] Combination therapy The method of the present invention also intends to use auxiliary active compounds and molecules in combination with transferrin and / or lactoferrin. These auxiliary active compounds and molecules can be formulated together with transferrin or lactoferrin as unit dosage forms, i.e., as physically distinct units intended as unit doses to the target to be treated. Alternatively, the auxiliary active compounds and molecules can be provided as a kit of parts: • Transferrin and / or lactoferrin are administered separately in a stepwise or sequential dosing pattern; or • They are administered simultaneously in different dosage forms.

[0055] For example, the methods of the present invention are intended to administer 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 suitable plasma sources such as human plasma, and those prepared using recombinant manufacturing techniques. For example, serum or plasma proteins may be selected from the group consisting of albumin (e.g., ALBUTEIN), alpha-1 antitrypsin / alpha-1 proteinase inhibitors (e.g., PROLASTIN), antithrombin (e.g., THROMBATE III), polyclonal immunoglobulins (IgG, IgA, and combinations thereof), polyspecific immunoglobulins (IgM), C1 esterase inhibitors (e.g., BERINERT), transthyretin, and combinations thereof.

[0056] Examples of 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%, and GAMMAGARD Liquid 10%.

[0057] Examples of 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 inhibitors, 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 a particular embodiment, a therapeutically effective dose of alpha-1 antitrypsin is administered to the patient in addition to a protein selected from transferrin, lactoferrin, and combinations thereof. In a particular embodiment, a therapeutically effective dose of antithrombin is administered to the patient in addition to a protein selected from transferrin, lactoferrin, and combinations thereof.

[0059] The present invention also provides a method for administering known neurogenesis / neurotrophic compounds and molecules in combination with transferrin and / or lactoferrin. For example, the present invention intends to administer neurogenesis / neurotrophic proteins, peptides, and small molecules together with transferrin and / or lactoferrin.

[0060] Appropriate neurogenesis 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 (ciliary 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 understand that the present invention also intends to include covalent conjugates of each of the compounds and molecules listed above to each of transferrin and lactoferrin. Furthermore, they will understand that the present invention also intends to include recombinant fusion proteins of each of the proteins and peptides listed above with each of transferrin and lactoferrin.

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

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

[0064] Pharmaceutical composition of the present invention In a further embodiment, the present invention also relates to novel neurological events in patients suffering from traumatic brain injury, non-traumatic brain injury, spinal cord injury, and combinations thereof. system The present invention provides a pharmaceutical composition containing transferrin, lactoferrin, or a combination thereof, for use in cell generation.

[0065] The pharmaceutical composition of the present invention may optionally further include at least one pharmaceutically acceptable carrier. The at least one pharmaceutically acceptable carrier may be selected from auxiliaries and vehicles. The at least one pharmaceutically acceptable carrier includes any solvent, diluent, other liquid vehicle, dispersing aid, suspension aid, surfactant, isotonic agent, thickener, emulsifier, and preservative suitable for a specific dosage form of the desired type.

[0066] Suitable carriers are described in Remington: The Science and Practice of Pharmacy, 21st edition, 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 (their contents are incorporated herein by reference). Preferred examples of such carriers or diluents, but not limited to, include water, physiological saline, Ringer's solution, glycol, dextrose solution, buffers (phosphates, glycine, sorbic acid, and potassium sorbate, etc.), and 5% human serum albumin. Liposomes and non-aqueous vehicles, such as glyceride mixtures of saturated vegetable fatty acids, and non-volatile oils (peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil, etc.) may also be used depending on the route of administration.

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

[0068] In one embodiment, parenteral administration is the preferred route of administration. The pharmaceutical composition may be sealed in glass or plastic ampoules, disposable syringes, sealed bags, or multi-dose vials. In one embodiment, intravenous injection is the preferred route of administration. The formulation can be administered continuously by drip infusion or by bolus injection.

[0069] The pharmaceutical composition of the present invention may be provided as a unit dosage form, that is, as physically distinct units intended as a unit dose to be administered to the target to be treated.

[0070] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (if water-soluble) or dispersants, and sterile powders for the immediate preparation of sterile injection solutions or dispersants. 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 fluid enough to allow easy passage through the injection needle.

[0071] The compositions of the present invention should be stable under manufacturing and storage conditions. Furthermore, the compositions should be protected against contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0072] Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars (e.g., mannitol, sorbitol), polyhydric alcohols, or sodium chloride in the composition. Sustained absorption of the injectable composition can be achieved by including an absorption-delaying agent in the composition, such as aluminum monostearate or gelatin.

[0073] A sterile injection solution of the pharmaceutical composition of the present invention can be prepared by incorporating the required amount of active molecules in a suitable solvent along with one or a combination of the components discussed above, followed by sterile filtration. In the case of a sterile powder for preparing a sterile injection solution, preparation methods include vacuum drying and freeze-drying, thereby obtaining a powder containing the active ingredient plus any desired additional components from a pre-sterilized filtered solution.

[0074] Unless any conventional medium or agent is incompatible with the active molecule of the present invention, its use in a composition is intended to be within the scope of the present invention.

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

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

[0077] dosage As discussed above, the inventors hypothesize that increasing the concentration of transferrin, lactoferrin, or combinations thereof proximal to the site of neurodegenerative injury or damage may 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 combinations thereof may be administered topically or proximal to damage caused by a neurodegenerative event by conventional routes selected from the group consisting of intracerebral, intracranial, intraspinal, and intrathecal. For example, transferrin, lactoferrin, and combinations thereof may be administered topically during surgical intervention.

[0078] Alternatively, transferrin, lactoferrin, or a combination thereof may 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, vaginal, and rectal administration.

[0079] To avoid any misunderstanding, let me clarify here that this specification discusses the iron saturation level of transferrin in two separate and distinct contexts: a) In the first context, as briefly stated herein, this specification describes 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 purified exogenous transferrin can be determined using inductively coupled plasma atomic emission spectroscopy (however, other methods such as colorimetric methods may also be used). b) In a second context which will be discussed in more detail shortly thereafter, this specification describes the measurement of the iron saturation of physiological transferrin in a patient, i.e., in the patient's plasma or serum, after the patient has been administered a pharmaceutical composition containing exogenous transferrin.

[0080] Under normal physiological conditions, substantially all iron in plasma is bound to transferrin, and the resulting physiological transferrin has an iron saturation of approximately 30%. In Example 6 (see below), the inventors demonstrated that unexpected nerve regeneration effects could be obtained with transferrin having an iron saturation of less than 30%. As a non-limiting hypothesis, it is assumed that administering a pharmaceutical composition containing exogenous transferrin (having low iron saturation) to a patient increases the physiological concentration of transferrin in the patient's plasma, resulting in a decrease in the iron saturation of physiological transferrin to less than 30%. Therefore, physiological transferrin becomes capable of utilizing nerve regeneration. 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] Therefore, in one embodiment, a protein selected from transferrin, lactoferrin, and combinations thereof is administered to the 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 combinations thereof is administered to the 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 combinations thereof may be administered to the 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 understand that measuring transferrin iron saturation levels in a patient's serum or plasma is a typical assay typically performed using the colorimetric methodology discussed above. The iron content of plasma or serum is measured by a chemical analyzer using a colorimetric reaction employing ferren or ferrosin as the dye source to form a color complex with iron. The sample analyzed yields two values: • The iron content of the sample (i.e., 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 and unincorporated iron-binding capacity (UIBC) of the sample. • Transferrin saturation (%) is determined as [(iron content of sample / TIBC) × 100].

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

[0084] In further embodiments of the method 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, for example, about 20 mg / kg to about 6000 mg / kg, for example, 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. Preferably, the protein can be administered at a concentration of about 50 mg / kg to about 500 mg / kg, for example, about 50 mg / kg to about 250 mg / kg, for example, about 50 mg / kg to about 150 mg / kg.

[0085] In one embodiment, the method of the present invention may include the step of administering a protein selected from transferrin, lactoferrin, and combinations thereof to a patient in need as part of a multi-dose regimen. For example, an initial dose of about 50 mg / kg to about 5000 mg / kg on day 1 of the administration period, followed by doses of about 50 mg / kg to about 1000 mg / kg per dose during the multi-dose period. For example, an initial dose of about 50 mg / kg to about 1000 mg / kg on day 1 of the administration period, followed by doses of about 50 mg / kg to about 500 mg / kg per dose during the multi-dose period. For example, an initial dose of about 50 mg / kg to about 500 mg / kg on day 1 of the administration period, followed by doses of about 50 mg / kg to about 250 mg / kg per dose during the multi-dose period. For example, an initial dose of about 50 mg / kg to about 250 mg / kg on day 1 of the administration period, followed by doses of about 50 mg / kg to about 250 mg / kg per dose during the multi-dose period. The period of multiple doses may include approximately 3 to 30 administrations up to the total cumulative dose. The period of multiple doses may also include approximately 1 to 30 weeks. Multiple partial doses may be administered at intervals of approximately 1 to 30 days.

[0086] Those skilled in the art will understand that the specific embodiments disclosed in the preceding paragraphs should not be read in isolation, and that this specification is intended to disclose these embodiments not individually, but in combination with other embodiments. For example, each of the embodiments disclosed in the preceding paragraphs should be read as being expressly combined with each of the embodiments in the preceding paragraphs, or with any two or more 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 drawing]

[0088] [Figure 1-1] Figure 1A is a graph showing the induction of neurite outgrowth in SH-SY5Y cells in response to apo-transferrin. 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 the increase in β-III-tubulin protein concentration in SH-SY5Y cells in response to apo-transferrin. Figure 1D is a graph showing the increase in β-III-tubulin protein concentration in SH-SY5Y cells in response to apo-transferrin. [Figure 2] This graph demonstrates 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 effects of various concentrations of deferoxamine mesylate on neurite outgrowth in SH-SY5Y cells relative to apo-transferrin. Figure 3B is a graph showing the efficacy of a transferrin mutant with reduced iron-binding capacity on promoting neurite outgrowth in SH-SY5Y cells. [Figure 4] This graph plots the effects of various different proteins on neurite outgrowth in SH-SY5Y cells. [Figure 5] This graph plots the effect of IOX2, a prolyl hydroxylase inhibitor, on neurite outgrowth in SH-SY5Y cells. [Figure 6] This graph shows the role of iron saturation in the efficacy of transferrin in promoting neurite outgrowth in SH-SY5Y cells. [Figure 7] This graph plots the effect of apo-transferrin, when used in combination with other neurotrophic protein / peptide fragments, on neurite outgrowth in SH-SY5Y cells. [Figure 8] This graph plots the effect of apo-transferrin in combination with the small molecule Y-27632 on neurite outgrowth in SH-SY5Y cells. [Figure 9]This graph shows that apo-transferrin increases the amount of neurogenesis when measured in newly formed neuroblasts (defined by BrdU+ / DCX+ cells) and newly formed mature neurons (defined by BrdU+ / NeuN+ cells) in animal test models. [Figure 10-1] Figure 10A is a graph demonstrating that animals treated with apo-transferrin after transient MCA administration recover faster compared to mice treated with saline. Figure 10B is a graph demonstrating that animals treated with apo-transferrin after transient MCA administration exhibit better motor skills compared to mice treated with saline. [Figure 10-2] Figure 10C is a graph demonstrating that animals treated with apo-transferrin after transient MCA administration exhibit higher cognitive abilities compared to mice treated with physiological saline. [Modes for carrying out the invention]

[0089] Detailed Examples of the Invention The examples disclosed herein are merely generalized embodiments, and it will be readily apparent to those skilled in the art that other configurations and methods capable of reproducing the present invention are possible and encompassed by the present invention. [Examples]

[0090] Apo-transferrin (ApoTf) dose-response induces differentiation and neurite outgrowth in SH-SY5Y cells. Transferrin is used 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, which is the transferrin receptor 1 (TfR1), and the subsequent endocytosis. Transferrin is typically thought to supply iron to cells as a means of promoting and sustaining metabolic activity. Surprisingly, we have found that apo-transferrin, a non-ferrous form of the transferrin protein, induces differentiation of SH-SY5Y cells, a very common neuronal research model. The induction of neuronal differentiation was evaluated by morphological parameters of neurite formation (key elements typically used as markers of neuronal differentiation, neuronal health, and function) following the procedures of Agholme, 2010. J. of Alzheimer's Disease. Vol. 20:1 pp. 069-108; 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 a medium containing 0.1% FBS. Serum-free basal medium was used as recommended by the SH-SY5Y cell supplier (Sigma, catalog number 94030304-1VL). Twenty-four hours after seeding, a 3× stock solution of ApoTf (final concentration in serum-free basal medium shown on the x-axis) was added to the cells. ApoTf was obtained from pooled human plasma, purified, and administered at a final concentration of 0.2 mg / mL. Cells were differentiated for 6 days. Neurite growth was evaluated by imaging and image analysis. At the time of analysis, 10× solutions of Tubulin Tracker (Molecular Probes, T34075) and Hoechst 33342 (Molecular Probes, #H3570) nuclear stains were prepared.

[0092] In short, Tubulin Tracker dissolved in DMSO was diluted 1:1 with Pluronic F-127, and then further diluted in HBSS to produce a 10× solution. Hoechst 33342 was added to the HBSS-Tubulin Tracker solution at a rate of 10 μg / mL to produce a 10× nuclear stain. This 10× stain solution (10 μL) was added directly to the processed 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 imaging system. For each image, nine images / well were acquired using the blue (nucleus) and green (tubulin) fluorescence channels.

[0093] After obtaining the images, the MetaExpress Neurite Outgrowth analysis module (Molecular Devices) was used to identify cells and cell bodies and quantify neurites. Considering the varying number of cells in each test well, the total number of neurite branching was divided by the total number of imaged cells. The magnification change in elongation was determined by setting the untreated control cells to a value of 1 and representing all other treatments relative to the untreated control.

[0094] Figure 1A clearly shows that apo-transferrin was able to induce neurite outgrowth in a dose-dependent manner. Gradual increases in apo-transferrin up to a maximum of 0.8 mg / mL enhanced the outgrowth response in SH-SY5Y cells. This phenomenon is counterintuitive to the known function of transferrin, which is mainly observed in holo- or iron-supported transferrins.

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

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

[0097] In addition, as shown in Figure 1D, treatment with apo-transferrin was found to induce an increase in 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, the cells were fixed with paraformaldehyde, stained for β-III-tubulin (R&D Systems, MAB1195), and imaged using a Molecular Devices Nano imaging system. Image analysis was performed by evaluating the fluorescence intensity of cells stained with β-III-tubulin. Background from a single secondary antibody was subtracted from all values. Values ​​under display conditions are shown as "β-III-tubulin staining intensity" along with the standard deviation.

[0098] SH-SY5Y cells "SH-SY5Y cells" in this specification refers to a subcloned cell line derived from the SK-N-SH neuroblastoma cell line. These cells can be converted to various types of functional nerves by adding specific compounds. system Because it can be converted into cells, it serves as a model for neurodegenerative disorders. In addition, the SH-SY5Y cell line is widely used in experimental neurological studies, including the analysis of neuronal differentiation, metabolism, and function related to neurodegenerative processes, neurotoxicity, and neuroprotection.

[0099] This specification briefly outlines, hereafter, peer-reviewed references that refer to the SH-SY5Y cell line as a predictive model for various neurodegenerative disorders. This list does not constitute an endorsement of prior art by the inventors, but rather serves to illustrate the knowledge of those skilled in the art regarding 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. October-December 2009; 29(4): pp. 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 (insecticides, chemical weapons compounds) Hong et al. Neurotoxicity induced in differentiated SK-N-SH-SY5Y human neuroblastoma cells by organophosphorus compounds. Toxicology and Applied Pharmacology 186 (2003) pages 110~118. 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):pages 51~63.

[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, pages 355~365. 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:pages 115~129. 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) pages 159~168. 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. [Examples]

[0107] The effect of ApoTf on β-III-tubulin and GFAP protein concentrations in primary human neural progenitor cells. The neurogenesis effects of ApoTf also apply to primary human cerebral cortex-derived neural progenitor cells, another established model of adult neurogenesis (see Azari and Reynolds, "In Vitro Models for neurogenesis," Cold Spring Harb Perspect 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 cells differentiated into astrocytes (GFAP-positive cells, 2B) from cultures of primary human cerebral cortex-derived neural progenitor cells compared to cells without apo-transferrin.

[0108] Neural progenitor cells maintained as neurospheres were obtained from Lonza (PT-2599). Cells were thawed from frozen neurosphere vials and cultured for 2 weeks in Human NeuroCult® NS-A Complete Proliferation medium (Stemcell Technologies). Neurospheres were isolated into single cells and plated into laminin-coated wells of assay plates. Neural progenitor cells were seeded for 72 hours in NeuroCult® NS-A basic medium containing 1 / 10th the recommended growth adjuvant concentration, either in the absence or in the presence of ApoTf (0.8 mg / mL). 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 imaging system. Image analysis was performed by evaluating the relative number of cells positively stained for β-III-tubulin or GFAP. The values ​​under the display conditions are shown as "β-III-tubulin positive %" cells (Figure 2A) or "GFAP positive %" cells (Figure 2B), along with their standard deviations. [Examples]

[0109] Iron chelation is not the only mechanism of action for neurogenesis by ApoTf. Deferoxamine mesylate (DFO) is a small molecule iron chelator used in the clinical management of iron overload. Similar to ApoTf, DFO has a high affinity binding constant for iron, but possesses only a single iron-binding site. The effect of DFO on neurite outgrowth was investigated. ApoTf was tested at concentrations close to the bottom of its functional dose curve and compared to the ability of DFO to induce neurite outgrowth. ApoTf tested at 2.4 μM (0.2 mg / mL) has two iron-binding sites, making it 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 growth was evaluated by imaging and image analysis. At the time of analysis, 10× solutions of Tubulin Tracker (Molecular Probes, T34075) and Hoechst 33342 (Molecular Probes, #H3570) nuclear stains were prepared. Briefly, Tubulin Tracker dissolved in DMSO was diluted 1:1 with Pluronic F-127 and further diluted in HBSS to produce a 10× solution. Hoechst 33342 was added to the HBSS-Tubulin Tracker solution at 10 μg / mL to produce a 10× nuclear stain. This 10× stain 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 imaging system. Nine images / well were acquired for each image using blue (nucleus) and green (tubulin) fluorescence channels. After image acquisition, cell bodies were identified and neurites were quantified using the MetaExpress Neurite Outgrowth analysis module (Molecular Devices). To account for the varying number of cells, the total number of neurite branching was divided by the total number of imaged cells. The magnitude change in elongation was determined by setting the untreated control cells to a value of 1 and showing all other treatments relative to the untreated control. ApoTf was obtained from pooled human plasma, purified, and administered at a final concentration of 0.2 mg / mL. Deferoxamine mesylate (DFO) was obtained from Tocris (catalog no. 5764), resuspended according to the manufacturer's recommendation, and stored. The DFO concentrations evaluated for neurogenesis are shown on the x-axis.

[0111] Figure 3 shows that DFO exhibits maximum neurite outgrowth between 1 and 3 μM concentrations, with almost no neurite formation occurring beyond this range, whereas ApoTf continues to increase differentiation even at 9.9 μM (0.8 mg / mL; 20 μM iron binding site). These data suggest that iron chelation may play a role in neurite outgrowth, but it is not the primary mechanism of action, and that other unidentified functional aspects of ApoTf must also play a role in its neurogenesis.

[0112] The inventors further sought to determine whether a reduction in transferrin's iron-binding activity due to mutations in the N-terminal iron-binding site is sufficient to mediate neurogenesis. Undifferentiated SH-SY5Y cells were treated as described in Example 1. Neurite growth was evaluated by imaging and image analysis. At the time of analysis, 10× solutions of Tubulin Tracker (Molecular Probes, T34075) and Hoechst 33342 (Molecular Probes, #H3570) nuclear stains were prepared. Briefly, Tubulin Tracker dissolved in DMSO was diluted 1:1 with Pluronic F-127 and further diluted in HBSS to produce a 10× solution. Hoechst 33342 was added to the HBSS-Tubulin Tracker solution at 10 μg / mL to produce a 10× nuclear stain. The 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 imaging system. For each image, nine images / well were acquired using blue (nucleus) and green (tubulin) fluorescence channels. After obtaining the images, cell bodies were identified and neurites were quantified using the MetaExpress Neurite Outgrowth analysis module (Molecular Devices).

[0113] To account for the differing cell counts, the total number of neurite branchings was divided by the total number of imaged cells. The magnification change in elongation was determined by setting the untreated control to a value of 1 and showing all other treatments relative to the untreated control. All proteins were administered 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] In short, 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 the proteins were collected from the cell culture supernatant. The proteins were purified using a Ni-NTA column, washed, and eluted. The N-terminal 6xHIS tag and additional amino acids were cleaved from the transferrin protein using TurboTEV protease. Following TEV cleavage, the transferrin protein was separated from the cleaved 6xHIS tag and the uncleaved protein using 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 residual iron that may be bound to these proteins. The buffer was replaced with PBS pH 7.4, the fraction was concentrated, and it was filtered to sterile for final use.

[0116] Figure 3B shows that plasma-derived human serum albumin (pdHSA) did not affect neurogenesis. However, both ApoTf and recombinant ApoTf induced SH-SY5Y neurogenesis. An ApoTf mutant with reduced iron-binding capacity (N-mut rec ApoTf) was almost equivalent to ApoTf and rec ApoTf in inducing SH-SY5Y cell differentiation. It appears that iron binding is not the only mechanism of action for ApoTf's neurogenesis potential. [Examples]

[0117] The neurogenesis effects on SH-SY5Y are specific to apo-transferrin and apo-lactoferrin. Since the role of iron chelation in the neurogenesis of ApoTf was found to be unclear from Example 3, the inventors determined whether other iron-binding proteins can also mediate neurogenesis in SH-SY5Y cells.

[0118] Undifferentiated SH-SY5Y cells were treated as described in Example 1. Neurite growth was evaluated by imaging and image analysis. At the time of analysis, 10× solutions of Tubulin Tracker (Molecular Probes, T34075) and Hoechst 33342 (Molecular Probes, #H3570) nuclear stains were prepared. Briefly, Tubulin Tracker dissolved in DMSO was diluted 1:1 with Pluronic F-127 and further diluted in HBSS to produce a 10× solution. Hoechst 33342 was added to the HBSS-Tubulin Tracker solution at 10 μg / mL to produce a 10× nuclear stain. This 10× 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 imaging system. Nine images / well were acquired for each image using blue (nucleus) and green (tubulin) fluorescence channels. After image acquisition, cell bodies were identified and neurites were quantified using the MetaExpress Neurite Outgrowth analysis module (Molecular Devices). To account for the varying number of cells, the total number of neurite branching was divided by the total number of imaged cells. The magnitude change in elongation was determined by setting the untreated control to a value of 1 and showing all other treatments relative to the untreated control. 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 administered at a final concentration of 0.2 mg / mL.

[0119] Figure 4 shows that neither bovine serum albumin (BSA) nor low-affinity iron-binding human serum albumin had any effect on neurogenesis. For further information on low-affinity iron-binding human serum albumin (rHSA), see Silva et al., 2009. Biochimica et Biophysica Acta, Vol. 1794, pp. 1449-1458. Holo-transferrin (HoloTf), an iron-saturated 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 supports the hypothesis that iron binding alone is not the mechanism of action for ApoTf's neurogenesis potential. Unexpectedly, apo-lactoferrin also induced differentiation of these cells. Apo-lactoferrin is a structural and functional congener of apo-transferrin, but is found in breast milk rather than plasma.

[0121] Apolactoferrin shares 61% identity with apolactoferrin, whereas apoferritin and human serum albumin (HSA) are structurally unrelated to either apolactoferrin or apolactoferrin. [Examples]

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

[0123] In demonstrating the premise that ApoTf mediates neurogenesis other than the HIF pathway, the inventors tested well-known, highly specific prolyl hydroxylase (PHD2) inhibitors 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. Vol. 8, p. 1488. IOX2 inhibits PHD2 with an IC50 of 22 nM. 50 It possesses the ability to induce upregulation of HIF-1α at low concentrations of about 1 μM in undifferentiated SH-SY5Y (Ross, US2016008437, see above).

[0124] Undifferentiated SH-SY5Y cells were seeded and treated as described in Example 1. Neurite growth was evaluated by imaging and image analysis. At the time of analysis, 10× solutions of Tubulin Tracker (Molecular Probes, T34075) and Hoechst 33342 (Molecular Probes, #H3570) nuclear stains were prepared. Briefly, Tubulin Tracker dissolved in DMSO was diluted 1:1 with Pluronic F-127 and further diluted in HBSS to produce a 10× solution. Hoechst 33342 was added to the HBSS-Tubulin Tracker solution at 10 μg / mL to produce a 10× nuclear stain. This 10× stain 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 imaging system. Nine images / well were acquired for each image using blue (nucleus) and green (tubulin) fluorescence channels. After image acquisition, cell bodies were identified and neurites were quantified using the MetaExpress Neurite Outgrowth analysis module (Molecular Devices). To account for the varying number of cells, the total number of neurite branching was divided by the total number of imaged cells. The magnitude change in elongation was determined by setting the untreated control to a value of 1 and showing all other treatments relative to the untreated control. ApoTf was obtained from pooled human plasma, purified, and administered at a final concentration of 0.2 mg / mL. IOX2 was obtained from Tocris (catalog no. 4451), resuspended according to the manufacturer's recommendation, and stored.

[0125] Figure 5 clearly shows that neither neurite outgrowth nor differentiation was observed in IOX2-treated cells. Even at an extremely high concentration of 4 μM of IOX2, no effect was observed (inducing HIF-1α in SH-SY5Y cells required a concentration four times higher than that reported in US2016008437, and Chowdhury showed IC12 for PHD2 protein). 50(More than 180 times higher than the concentration determined). When these data are combined with the lack of neurogenesis in HoloTf (Example 4), it is shown that HIF-1α does not play a role in the differentiation of SH-SY5Y cells. [Examples]

[0126] The role of iron saturation in the efficacy of transferrin ApoTf samples with various purities and iron saturations, as briefly described in Table 1, were evaluated for their neurogenesis potential. Transferrin samples were prepared according to procedures / methodologies known to those skilled in the art. These are described in detail in L von Bonsdorff et al., Transferrin, Chapter 21, Section 21.4, pp. 301-310, and Production of Plasma Proteins for Therapeutic Use, edited by J. Bertolini et al., Wiley, 2013 [Print ISBN: 9780470924310 | Online ISBN: 9781118356807] (their contents 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 growth was evaluated by imaging and image analysis. At the time of analysis, 10× solutions of Tubulin Tracker (Molecular Probes, T34075) and Hoechst 33342 (Molecular Probes, #H3570) nuclear stains were prepared. Briefly, Tubulin Tracker dissolved in DMSO was diluted 1:1 with Pluronic F-127 and further diluted in HBSS to produce a 10× solution. Hoechst 33342 was added to the HBSS-Tubulin Tracker solution at 10 μg / mL to produce a 10× nuclear stain. This 10× 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 imaging system. Nine images / well were acquired for each image using blue (nucleus) and green (tubulin) fluorescence channels. After image acquisition, cell bodies were identified and neurites were quantified using the MetaExpress Neurite Outgrowth analysis module (Molecular Devices). To account for the varying number of cells, the total number of neurite branching was divided by the total number of imaged cells. The magnitude change in elongation was determined by setting the untreated control to a value of 1 and showing all other treatments relative to the untreated control.

[0130] Figure 6A plots the effects of ApoTf A-D, administered at a final concentration of 0.2 mg / mL, with the purity and iron content described in Table 1, on neurite outgrowth in SH-SY5Y cells. Figure 6B plots the effects of transferrin with various iron saturation levels (listed on the X axis), administered 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 briefly described by von Bonsdorff (see above). By mixing ApoTf and HoloTf, various iron saturation contents were generated to produce the displayed saturation percentages plotted in Figure 6B.

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

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

[0134] The function of proteins from three neurotrophic superfamilies in combination with ApoTf was investigated. 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, the function of PACAP (amino acids 1-38 of pituitary adenylyl cyclase-activating polypeptide), another known neurotrophic peptide, in combination with ApoTf was evaluated.

[0135] Undifferentiated SH-SY5Y cells were treated as described in Example 1. Neurite growth was evaluated by imaging and image analysis. At the time of analysis, 10× solutions of Tubulin Tracker (Molecular Probes, T34075) and Hoechst 33342 (Molecular Probes, #H3570) nuclear stains were prepared. Briefly, Tubulin Tracker dissolved in DMSO was diluted 1:1 with Pluronic F-127 and further diluted in HBSS to produce a 10× solution. Hoechst 33342 was added to the HBSS-Tubulin Tracker solution at 10 μg / mL to produce a 10× nuclear stain. This 10× 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 imaging system. Nine images / well were acquired for each image using blue (nucleus) and green (tubulin) fluorescence channels. After image acquisition, cell bodies were identified and neurites were quantified using the MetaExpress Neurite Outgrowth analysis module (Molecular Devices). To account for the varying number of cells, the total number of neurite branching was divided by the total number of imaged cells. The magnitude change in elongation was determined by setting the untreated control to a value of 1 and showing all other treatments relative to the untreated control.

[0136] In Figures 7A-7D, ApoTf was administered 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 number 450-02) and administered at 25 ng / mL. (B) GDNF was obtained from Peprotech (catalog number 450-10) and administered at 1000 ng / mL. (C) CNTF was obtained from Peprotech (catalog number 450-13) and administered at 250 ng / mL. (D) PACAP was obtained from Tocris (catalog number 1186) and administered at 200 nM. The abbreviation SF represents serum-free medium.

[0137] A review of Figures 7A-7D clearly shows that each neurotrophic factor and peptide fragment induced SH-SY5Y cell differentiation to varying degrees. In some cases, such as with BDNF, differentiation was not induced at the tested concentrations of neurotrophic factors in the absence of ApoTf. In all of the experiments presented, neurotrophic factors used in combination with ApoTf induced more differentiation than those tested alone. Unexpectedly, ApoTf exhibits synergistic effects with other neurotrophic factors and peptides on neurite outgrowth in SH-SY5Y cells. [Examples]

[0138] Apo-transferrin acts synergistically with small neurons to induce differentiation. The ability of ApoTf to act in combination with non-protein-based neurogenesis small molecule compounds was tested in Example 7. ApoTf was evaluated in combination with the neurogenesis compound Y-27632 [trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride]. Y-27632 is a Rock1 and Rock2 (Rho kinase) inhibitor. Inhibition of Rock1 and Rock2 by small molecules has a known ability to induce neuronal differentiation, including SH-SY5Y cells. See Dyberg et al., 2017. PNAS Vol. 114(32), E6603-E6612.

[0139] Undifferentiated SH-SY5Y cells were treated as described in Example 1. Neurite growth was evaluated by imaging and image analysis. At the time of analysis, 10× solutions of Tubulin Tracker (Molecular Probes, T34075) and Hoechst 33342 (Molecular Probes, #H3570) nuclear stains were prepared. Briefly, Tubulin Tracker dissolved in DMSO was diluted 1:1 with Pluronic F-127 and further diluted in HBSS to produce a 10× solution. Hoechst 33342 was added to the HBSS-Tubulin Tracker solution at 10 μg / mL to produce a 10× nuclear stain. This 10× 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 imaging system. Nine images / well were acquired for each image using blue (nucleus) and green (tubulin) fluorescence channels. After image acquisition, cell bodies were identified and neurites were quantified using the MetaExpress Neurite Outgrowth analysis module (Molecular Devices). To account for the varying number of cells, the total number of neurite branching was divided by the total number of imaged cells. The magnitude change in elongation was determined by setting the untreated control to a value of 1 and showing all other treatments relative to the untreated control. ApoTf was administered at a final concentration of 0.1 mg / mL, either alone or in combination with the indicated small molecule. Y-27632 was obtained from Tocris (catalog number 1254) and administered at 50 μM.

[0140] Figure 8 shows that while Y-27632 itself is a strong neurogenic compound, its neurogenic effect is synergistic in the presence of ApoTf, exceeding the effects of either molecule alone. The ability of ApoTf to act synergistically with several known proteins, peptides, and small molecule neurogenic entities is an unexpected, surprising, and intriguing discovery. [Examples]

[0141] Apo-transferrin promotes the formation of new neuroblasts and mature neurons in the brains of animals with transient MCAo. C57BL / 6J mice (approximately 20g) were anesthetized under isoflurane, and after incision, 6.0 silicone-coated monofilament sutures were inserted into the external carotid artery to occlude the middle cerebral artery (MCAo). Occlusion was performed for 60 minutes under temperature control. Within two hours of deocclusion, the animals were evaluated on a 7-point "neuroscore" scale to identify candidates for visually indicating stroke. This scale ranged from 0 (no observable loss) to 6 (near-fatal), taking into account contralateral forelimb extension, severity of turning, gait, and loss of consciousness. 0 = No observable defects 1 = Impairment of extension of the contralateral forelimb 2 = Slight turning behavior when the tail is lifted, a <50% tendency to rotate to the opposite side. 3 = Mild, unchanging turning behavior; >50% tendency to rotate to the opposite side. 4 = Unchanging, strong turning; the mouse maintains the rotating position for longer than 1-2 seconds, and its nose almost touches its tail. 5 = Severe rotational, gait, or righting reflex accompanied by a fall to the opposite side of the infarction, 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 for a total of 7 days with either 350 mg / kg of apo-transferrin (intraperitoneal injection) or the same amount of saline, along with 50 mg / kg of bromodeoxyuridine (BrdU; Sigma-Aldrich Chemical, St. Louis, MO). Both were delivered intraperitoneally. Body weight changes were monitored daily.

[0143] At the point shown in Example 9, the brain was prepared for analysis by transcardiac perfusion with ice-cold heparinized saline (2.5 IU / ml heparin) to remove blood from the brain. The fresh brain was removed with the right cerebellum attached to the entire left hemisphere to help locate the brain block during dissection. The entire left hemisphere block was placed in 0.1 M phosphate buffer (PB) containing 4% paraformaldehyde at +4°C for 24 hours, and then cryoprotected on a shaker at +4°C for 2-3 days in 0.1 M PB containing 30% sucrose. The brain was then blotted to remove excess fluid, placed on the cork of a vial, and frozen over liquid nitrogen. This block was then stored at -80°C until cryostat sections were obtained. Neurogenesis was assessed by immunohistochemical tests using antibodies against BrdU and double cortin (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 administration of ApoTf increases the number of neuroblasts over a two-week period, while Figure 9B shows that after four weeks, ApoTf-treated mice have a greater number of newly formed mature neurons. These data suggest that neuroblasts formed in the early stages of neurogenesis can continue to differentiate and become even more new mature neurons. Therefore, these results suggest that apo-transferrin can promote aspects of neurogenesis beyond those normally induced in response to ischemic stroke. [Examples]

[0145] Apo-transferrin promotes recovery, motor skills, and cognitive function in a mouse model of transient MCAo stroke. Mice were prepared as shown in Example 9 above and evaluated as shown 3 days after MCAo and at 1, 2, 3, and 4 weeks (n=8-10 animals / group). Motor coordination (i.e., balance behavior and gait ability in accordance with the function of the corticostriate system) was evaluated using the rotarod test. Learning and memory abilities were 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 of recovery in animals after MCA occlusion (MCAo) over a period of 3–14 days, and possibly longer. As described in Example 9, all animals in this study had an initial neuronal score of 4 or higher when assessed 2 hours after MCAo. The percentage of animals with no observable defects (i.e., neuronal scores of "0" as described in Example 9) is shown as a function of time after MCAo on the x-axis.

[0147] Figure 10B shows that animals treated with apo-transferrin after MCAo exhibit enhanced motor and balance skills. The time it takes for an animal to fall from the rotorod (latency to fall) is shown relative to the time after MCAo. The motor skills of the mice reflect those of the neural scores in Figure 10A, and the rate of improvement in motor / balance skills is increased when measured by the time the animals remained on the rotorod device. Recovery, as measured by neural scores, was the same in both groups by 4 weeks, but mice treated with ApoTf had a better overall ability to remain on the rotorod. Figure 10C provides evidence that administration of ApoTf enhances learning and cognitive function for at least 2 weeks after protein administration. Discriminative ability (%) represents the animal's memory and is measured as the percentage of time the animal spends examining a newly presented object compared to the time it spends examining a previously presented object. Animals with better cognitive ability and memory spend more time on new objects because they have memory of the previously presented objects. This is shown as an increased percentage of discriminative ability. Since animals treated with apoTf have a higher "percentage of discriminative ability" compared to mice treated with physiological saline, it is suggested that mice treated with apoTf after MCAo recover their cognitive abilities more effectively.

[0148] In summary, the data from Figures 10A to 10C suggest that neurogenesis promoted by apoTf leads to better motor and cognitive abilities in the subjects.

[0149] array The sequences mentioned in the preceding paragraph are briefly described below in fasta format.

[0150] Sequence ID 1: Human transferrin [UniProt Q06AH7] protein sequence

[0151] [ka]

[0152] Sequence ID 2: Human lactoferrin [UniProt P02788] protein sequence

[0153] [ka]

[0154] Sequence ID 3: Y188F transferrin N-lobe mutant protein

[0155] [ka]

[0156] Sequence ID 4: Y95F / Y188F transferrin N-lobe mutant protein

[0157] [ka]

[0158] Sequence ID 5: Y426F / Y517F transferrin C-lobe mutant protein

[0159] [ka]

[0160] Sequence ID 6: BDNF

[0161] [ka]

[0162] Sequence ID 7: GDNF

[0163] [ka]

[0164] Sequence ID 8: CNTF

[0165] [ka]

[0166] Sequence ID 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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