Compositions for nerve regeneration

Abstract

Disclosed herein is a pharmaceutical composition containing transferrin or lactoferrin for use in promoting or inducing the generation of new nerve cells in patients suffering from neurodegenerative events. Neurodegenerative events may be caused by neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis. Ideally, the transferrin and / or lactoferrin has low iron saturation.

Description

Technical Field

[0001] The present invention relates to therapeutic proteins and use thereof in the field of regenerative medicine. In particular, disclosed herein are transferrin and lactoferrin, and use thereof in promoting the proliferation, induction and / or differentiation of neural progenitor cells or neural stem cells.

Background Art

[0002] Neurodegenerative diseases such as amyotrophic lateral sclerosis, Huntington's disease, Alzheimer's disease, and Parkinson's disease are conditions characterized by progressive neuronal cell death, and are associated with high morbidity, patient suffering, reduced quality of life, and high mortality. As demographics gradually shift to an aging population, the prevalence of neurodegenerative conditions in society has risen sharply. Life expectancy continues to increase, and it is expected that neurodegenerative conditions will stand alongside cancer and cardiovascular diseases as the leading causes of death for future generations.

[0003] As of the time of writing, without exception, there are no approved therapies capable of curing or reversing the effects of neurodegenerative diseases. In certain specific situations, there are approved drugs that delay disease progression. For example, riluzole (RILUTEK) and edaravone (RADICAVA) are two approved therapies for the treatment of amyotrophic lateral sclerosis that slow disease progression, but said molecules do not reverse the symptoms of the disease once it has onset.

[0004] Given the absence of curative therapies, it is not surprising that most commercially approved therapies focus on symptomatic treatment, including, inter alia, dopaminergic treatments for Parkinson's disease and movement disorders, antipsychotics for behavioral and psychological symptoms of dementia, and analgesics for pain management.

[0005] Neuroprotection is an alternative, non-recoverable approach to managing neurodegenerative diseases. Numerous reports in scientific and patent literature describe neuroprotective compounds and molecules that aim to limit damage caused by neurodegenerative diseases and slow their progression.

[0006] One such example is U.S. Patent Publication No. 2016008437, published by Grifols Worldwide Operations Ltd, which discloses a mixture of apo-transferrin and holo-transferrin that exerts neuroprotective effects by modulating the activity of hypoxia-inducible factor (HIF) in several degenerative disease conditions. Similarly, International Patent Application Publication No. WO2006 / 20727 by HealthPartners Research Foundation proposes the use of dephoroxamine as a modulator of hypoxia-inducible factor-1α to induce a neuroprotective response against the adverse effects of reperfusion in ischemic patients. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] U.S. Patent Publication No. 2016008437 [Patent Document 2] International Patent Application Publication Number WO2006 / 20727 [Non-patent literature]

[0008] [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] Yuan et al. The Mood Stabilizer Valproic Acid Activates Mitogen-activated Protein Kinases and Promotes Neurite Growth. JBC Vol. 276, No. 34, published August 24, pp. 31674-31683, 2001. [Non-Patent Document 15] Tatro et al. Modulation of Glucocorticoid Receptor Nuclear Translocation in Neurons by Immunophilins FKBP51 and FKBP52: Implications for Major Depressive Disorder. Brain Res. August 25, 2009; 1286: pp. 1-12. [Non-Patent Document 16] Laifenfeld et al. Norepinephrine alters the expression of genes involved in neuronal sprouting and differentiation: relevance for major depression and antidepressant mechanisms. Journal of Neurochemistry, 2002, 83, pp. 1054-1064 [Non-Patent Document 17] Cavarec et al. In Vitro Screening for Drug-Induced Depression and / or Suicidal Adverse Effects: A New Toxicogenomic Assay Based on CE-SSCP Analysis of HTR2C mRNA Editing in SH-SY5Y Cells. Neurotoxicity Research. January 2013, Vol. 23, No. 1, pp. 49-62. [Non-Patent Document 18] Jamsa et al. The retinoic acid and brain-derived neurotrophic factor differentiated SH-SY5Y cell line as a model for Alzheimer's disease-like tau phosphorylation. Biochemical and Biophysical Research Communications 319 (2004) pp. 993-1000 [Non-Patent Document 19] Seidel et al. Induced Tauopathy in a Novel 3D-Culture Model Mediates Neurodegenerative Processes: A Real-Time Study on Biochips. PLOS One. (November 2012) Vol. 7, No. 11. e49150 [Non-Patent Document 20] Karch et al., *Extracellular Tau Levels Are Influenced by Variability in Tau That Is Associated with Tauopathies*. JBC, Vol. 287, Vol. 51, pp. 42751-42762, December 14, 2012. [Non-Patent Document 21] Pettifer et al. Guanosine protects SH-SY5Ycells against b-amyloid-induced apoptosis. NeuroReport 2004 15(5):833~836 [Non-Patent Document 22] Tanii et al. Alzheimer's Disease: Presenilin-1 Exon 9 Deletion and L250s Mutations Sensitize SH-SY5Y Neuroblastoma Cells To Hyperosmotic Stress-Induced Apoptosis. Neuroscience, Vol. 95, No. 2, pp. 593-601, 2000. [Non-Patent Document 23] Li et al. Beta-amyloid induces apoptosis in human-derived neurotypic SH-SY5Y cells. Brain Res. November 4, 1996; 738(2): pp. 196-204. [Non-Patent Document 24] Lee et al. Hexanucleotide Repeats in ALS / FTD Form Length-Dependent RNA Foci, Sequester RNA Binding Proteins, and Are Neurotoxic. Cell Reports 5, pp. 1178-1186, December 12, 2013 [Non-Patent Document 25] Farg et al. C9ORF72, implicated in amyotrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking. Human Molecular Genetics, 2014, Volume 23, Issue 13 [Non-Patent Document 26] Nonaka et al. Phosphorylated and ubiquitinated TDP-43 pathological inclusions in ALS and FTLD-U are recapitulated in SH-SY5Y cells. FEBS Letters 583 (2009) pp. 394-400 [Non-Patent Document 27] Xing et al. Protective effects and mechanisms of Ndfip1 on SH-SY5Y cell apoptosis in an in vitro Parkinson's disease model. Genetics and Molecular Research 15 (2): gmr.15026963 [Non-Patent Document 28] Jung et al. Rosiglitazone protects human neuroblastoma SH-SY5Y cells against MPP+ induced cytotoxicity via inhibition of mitochondrial dysfunction and ROS production. Journal of the Neurological Sciences 253 (2007) pp. 53-60 [Non-Patent Document 29] Choi et al. Signaling Pathway Analysis of MPP+-treated Human Neuroblastoma SH-SY5Y Cells. Biotechnology and Bioprocess Engineering 19: pp. 332-340 (2014) [Non-Patent Document 30] Palomo et al. Silencing of frataxin gene expression triggers p53-.dependent apoptosis in human neuron-like cells. Human Molecular Genetics, 2011, Vol. 20, No. 14, pp. 2807-2822. [Non-Patent Document 31] Banez-Coronel et al. A Pathogenic Mechanism in Huntington's Disease Involves Small CAG-Repeated RNAs with Neurotoxic Activity. Neuroscience Research, Vol. 53, No. 3, November 2005, pp. 241-249. [Non-Patent Document 32] Vidoni et al. Resveratrol protects neuronal-like cells expressing mutant Huntingtin from dopamine toxicity by rescuing ATG4-mediated autophagosome formation. Neurochemistry International 117 (2018) pp. 174-187 [Non-Patent Document 33] Vidoni et al. Dopamine exacerbates mutant Huntingtin toxicity via oxidative mediated inhibition of autophagy in SH-SY5Y neuroblastoma cells: Beneficial effects of anti-oxidant therapeutics. Neurochemistry International 101 (2016) pp. 132-143 [Non-Patent Document 34] Olsen et al. Examination of mesenchymal stem cell-mediated RNAi transfer to Huntington's disease affected neuronal cells for reduction of huntingtin. Molecular and Cellular Neuroscience 49 (2012) pp. 271-281 [Non-Patent Document 35] Azari and Reynolds, “In Vitro Models for neurogenesis”. Cold Spring Harb Perspect Biol 2016, 8, a021279 [Non-Patent Document 36] Silva et al., 2009. Biochimica et Biophysica Acta, Vol. 1794, pp. 1449-1458. [Non-Patent Document 37] Chowdhury et al., 2013. ACS Chem. Biol. Vol. 8, p. 1488. [Non-Patent Document 38] Houlton et al., 2019. Frontiers in Neurosci., Vol. 13, Article 790. [Non-Patent Document 39] Weissmiller and Wu, 2012. Translational Neurodegeneration, Vol. 1: 14 [Non-Patent Document 40] Apfel, 2001. Clin Chem Lab Med., Vol. 39(4), p. 351. [Non-Patent Document 41] Sedelis et al., Behavioural Brain Research 125 (2001), pp. 109-122. [Non-Patent Document 42] Przedborski and Vila, Clinical Neuroscience Research 1 (2001), pp. 407-418. [Overview of the project] [Problems that the invention aims to solve]

[0009] Despite the above, it is clear that there are insufficient clinical candidates with the potential to cure or reverse the debilitating effects of neurodegenerative diseases and conditions. Approved clinical therapies are limited to addressing the symptoms of the disease, and the need for treatments that go beyond neuroprotection and slowing the progression of the condition through pathways remains unmet. Innovative technologies capable of curing or at least partially reversing nerve damage have yet to be realized and are therefore highly desirable. [Means for solving the problem]

[0010] 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.

[0011] 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.

[0012] 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.

[0013] Treatment method In a first aspect, the present invention relates to new nerves in patients suffering from neurodegenerative events. systemThe present invention provides a method for promoting and / or inducing cell generation, comprising the step of administering a therapeutically effective amount of a protein selected from transferrin, lactoferrin, and combinations thereof to a patient in need thereof.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] "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...

[0018] 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%.

[0019] 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.

[0020] 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.

[0021] 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).

[0022] 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.

[0023] 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).

[0024] 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.

[0025] "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...

[0026] 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%.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] In further embodiments, the present invention includes specific mutant transferrins and / or lactoferrins that maintain their structure but do not bind iron to the iron-binding domain, for example, one or both of the N-lobe and C-lobe, or a combination thereof.

[0032] 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).

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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 are extremely effective in stimulating cell development. Therefore, this invention relates to the role of nerves in patients suffering from neurodegenerative events. system The present invention provides a method for stimulating cell development, comprising the step of administering a therapeutically effective amount of a protein selected from transferrin, lactoferrin, and combinations thereof to a patient in need thereof.

[0041] When used herein, "nerve" system The term "stimulating cell development" means that transferrin or lactoferrin stimulates the development of new nerve cells. systemIt 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.

[0042] "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.

[0043] 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 involves 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.

[0044] In one embodiment, neurodegenerative events are caused by neurodegenerative diseases. “Neurodegenerative disease” means any disease characterized by dysfunction and / or death of neurons leading to loss of neuronal function in the brain, spinal cord, central nervous system, and / or peripheral nervous system. Neurodegenerative diseases within the scope of the present invention may be chronic or acute.

[0045] Non-limiting examples of neurodegenerative diseases within the scope of the present invention include Parkinson's disease, frontotemporal dementia, Alzheimer's disease, mild cognitive impairment, diffuse Lewy body dementia, Lewy body dementia, demyelinating diseases such as multiple sclerosis and acute transverse myelitis, amyotrophic lateral sclerosis, Huntington's disease, Creutzfeldt-Jakob disease, corticobasal ganglia ganglion degeneration, peripheral neuropathy, progressive supranuclear palsy, spinocerebellar degeneration, spinal ataxia, Friedreich's ataxia, cerebellar cortical degeneration, neurogenic muscular atrophy, anterior horn cell degeneration, infantile spinal muscular atrophy and juvenile spinal muscular atrophy, subacute sclerosing panencephalitis, Haller-Holden-Spats disease, boxer's dementia, Pick's disease, tauopathy, synuclein disease, and combinations thereof.

[0046] In one embodiment, the neurodegenerative disease may be selected from the group consisting of Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, and Huntington's disease. For example, the neurodegenerative disease may be Parkinson's disease.

[0047] 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.

[0048] 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 disclosed embodiments should be read as explicitly combined with each of the embodiments, or with any two or more of the embodiments disclosed herein in any order.

[0049] 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.

[0050] 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.

[0051] 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%.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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 disclosed embodiments should be read as explicitly combined with each of the embodiments, or with any two or more of the embodiments disclosed herein in any order.

[0059] Pharmaceutical composition of the present invention In a further embodiment, the present invention also relates to novel nerves in patients suffering from neurodegenerative events. system The present invention provides a pharmaceutical composition containing transferrin, lactoferrin, or a combination thereof, for use in cell generation.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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 disclosed embodiments should be read as explicitly combined with each of the embodiments, or with any two or more of the embodiments disclosed herein in any order.

[0072] 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.

[0073] 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.

[0074] To avoid any misunderstanding, let me clarify here that this specification discusses the iron saturation levels 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.

[0075] 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%.

[0076] 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.

[0077] 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].

[0078] 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).

[0079] 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.

[0080] 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.

[0081] 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 disclosed embodiments should be read as explicitly combined with each of the embodiments, or with any two or more of the embodiments disclosed herein in any order.

[0082] Additional features and advantages of the present invention will become more apparent in the accompanying drawings. [Brief explanation of the drawing]

[0083] [Figure 1AB] Figure 1A: Graph showing the induction of neurite outgrowth in SH-SY5Y cells in response to apo-transferrin. Figure 1B: Graph showing the induction of proliferation in SH-SY5Y cells in response to apo-transferrin. [Figure 1CD] Figure 1C: Image showing the increase in β-III-tubulin protein concentration in SH-SY5Y cells in response to apo-transferrin. Figure 1D: 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 3A] This graph plots the effects of various concentrations of deferoxamine mesylate on neurite outgrowth in SH-SY5Y cells relative to apo-transferrin. [Figure 3B] This graph shows the efficacy of a transferrin mutant with reduced iron-binding capacity in 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 demonstrates the positive regenerative effect of apo-transferrin in MPTP-induced Parkinson's disease in mice. [Modes for carrying out the invention]

[0084] 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]

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] This specification briefly outlines the 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 usefulness of the SH-SY5Y cell line as a predictive model for neurodegenerative disorders.

[0095] 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.

[0096] Mood stabilization (depression) Yuan et al. The Mood Stabilizer Valproic Acid Activates Mitogen-activated Protein Kinases and Promotes Neurite Growth. JBC Vol. 276, No. 34, published August 24, pp. 31674-31683, 2001. Tatro et al. Modulation of Glucocorticoid Receptor Nuclear Translocation in Neurons by Immunophilins FKBP51 and FKBP52: Implications for Major Depressive Disorder. Brain Res. August 25, 2009; 1286: pp. 1-12. Laifenfeld et al. Norepinephrine alters the expression of genes involved in neuronal sprouting and differentiation: relevance for major depression and antidepressant mechanisms. Journal of Neurochemistry, 2002, 83, pp. 1054-1064. Cavarec et al. In Vitro Screening for Drug-Induced Depression and / or Suicidal Adverse Effects: A New Toxicogenomic Assay Based on CE-SSCP Analysis of HTR2C mRNA Editing in SH-SY5Y Cells. Neurotoxicity Research. January 2013, Vol. 23, No. 1, pp. 49-62.

[0097] Tauopathy (Alzheimer's disease, FTD, and other neurodegenerative diseases involving abnormal tau) Jamsa et al. The retinoic acid and brain-derived neurotrophic factor differentiated SH-SY5Y cell line as a model for Alzheimer's disease-like tau phosphorylation. Biochemical and Biophysical Research Communications 319 (2004) pp. 993-1000. Seidel et al. Induced Tauopathy in a Novel 3D-Culture Model Mediates Neurodegenerative Processes: A Real-Time Study on Biochips. PLOS One. (November 2012) Vol. 7, No. 11. e49150. Karch et al. Extracellular Tau Levels Are Influenced by Variability in Tau That Is Associated with Tauopathies. JBC Vol. 287, Vol. 51, pp. 42751-42762, December 14, 2012.

[0098] Alzheimer's disease Pettifer et al. Guanosine protects SH-SY5Y cells against b-amyloid-induced apoptosis. NeuroReport 2004 15(5):833-836. Tanii et al. Alzheimer's Disease Presenilin-1 Exon 9 Deletion And L250s Mutations Sensitize SH-SY5Y Neuroblastoma Cells To Hyperosmotic Stress-Induced Apoptosis. Neuroscience, Vol. 95, No. 2, pp. 593-601, 2000. Li et al. Beta-amyloid induces apoptosis in human-derived neurotypic SH-SY5Y cells. Brain Res. November 4, 1996; 738(2): pp. 196-204.

[0099] ALS and frontotemporal dementia Lee et al. Hexanucleotide Repeats in ALS / FTD Form: Length-Dependent RNA Foci, Sequester RNA Binding Proteins, and Are Neurotoxic. Cell Reports 5, pp. 1178-1186, December 12, 2013. Farg et al. C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking. Human Molecular Genetics, 2014, Volume 23, Issue 13. Nonaka et al. Phosphorylated and ubiquitinated TDP-43 pathological inclusions in ALS and FTLD-U are recapitulated in SH-SY5Y cells. FEBS Letters 583 (2009) pp. 394-400.

[0100] Parkinson's disease Xing et al. Protective effects and mechanisms of Ndfipl on SH-SY5Y cell apoptosis in an in vitro Parkinson's disease model. Genetics and Molecular Research 15 (2): gmr.15026963. Jung et al. Rosiglitazone protects human neuroblastoma SH-SY5Y cells against MPP+ induced cytotoxicity via inhibition of mitochondrial dysfunction and ROS production. Journal of the Neurological Sciences 253 (2007) pp. 53-60. Choi et al. Signaling Pathway Analysis of MPP+-treated Human Neuroblastoma SH-SY5Y Cells. Biotechnology and Bioprocess Engineering 19: pp. 332-340 (2014).

[0101] Friedreich's ataxia Palomo et al. Silencing of frataxin gene expression triggers p53-.dependent apoptosis in human neuron-like cells. Human Molecular Genetics, 2011, Vol. 20, No. 14, pp. 2807-2822.

[0102] Huntington's disease Banez-Coronel et al. A Pathogenic Mechanism in Huntington's Disease Involves Small CAG-Repeated RNAs with Neurotoxic Activity. Neuroscience Research, Vol. 53, No. 3, November 2005, pp. 241-249. Vidoni et al. Resveratrol protects neuronal-like cells expressing mutant Huntingtin from dopamine toxicity by rescuing ATG4-mediated autophagosome formation. Neurochemistry International 117 (2018) pp. 174-187. Vidoni et al. Dopamine exacerbates mutant Huntingtin toxicity via oxidative mediated inhibition of autophagy in SH-SY5Y neuroblastoma cells: Beneficial effects of anti-oxidant therapeutics. Neurochemistry International 101 (2016) pp. 132-143. Olsen et al. Examination of mesenchymal stem cell-mediated RNAi transfer to Huntington's disease affected neuronal cells for reduction of huntingtin. Molecular and Cellular Neuroscience 49 (2012) pp. 271-281. [Examples]

[0103] 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.

[0104] 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]

[0105] 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.

[0106] 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.

[0107] Figure 3A 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.

[0108] The inventors further sought to determine whether a reduction in the iron-binding activity of transferrin due to a mutation in the N-terminal iron-binding site is sufficient to mediate neurogenesis.

[0109] 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. All proteins were administered at a final concentration of 0.2 mg / mL.

[0110] 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.

[0111] 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.

[0112] 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]

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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 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.

[0117] Apolactoferrin shares 61% identity with apolactoferrin, whereas apoferritin and human serum albumin (HSA) are structurally unrelated to either apolactoferrin or apolactoferrin. [Examples]

[0118] 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.

[0119] 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).

[0120] 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.

[0121] 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]

[0122] 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).

[0123] 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).

[0124] [Table 1]

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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]

[0129] 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.

[0130] 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 the pituitary adenylyl cyclase-activating polypeptide), another known neurotrophic peptide, in combination with ApoTf was evaluated.

[0131] 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.

[0132] 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.

[0133] 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]

[0134] 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.

[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. 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.

[0136] 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 and surprising discovery. [Examples]

[0137] Improvement of walking and movement in a mouse model of Parkinson's disease by apo-transferrin treatment. To illustrate that the above in vitro results can successfully translate into positive clinical effects, the inventors conducted a demonstration experiment of this therapy in a mouse model of Parkinson's disease. Mice were administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to destroy dopaminergic neurons in the substantia nigra, inducing Parkinson's disease in the mice. For further details, see Sedelis et al., Behavioural Brain Research 125 (2001), pp. 109-122; Przedborski and Vila, Clinical Neuroscience Research 1 (2001), pp. 407-418.

[0138] Destruction of dopaminergic neurons adversely affects animal movement. Mouse movement and gait can be measured by video analysis. As shown in Figure 9, substantial changes in movement and gait were observed in mice exposed to MPTP compared to control mice (n=15). Consistent with the findings in Examples 1-8, MPTP-induced Parkinson's disease in mice was significantly improved by administration of ApoTf (n=15). Figure 9 demonstrates the neuronal regenerative properties of ApoTf by showing that it significantly improved motor dysfunction in affected mice and effectively normalized the mice to the control level.

[0139] The animal experiments were conducted at Charles River Laboratories (Finland) in accordance with the guidelines of the National Institute of Health (Bethesda, MD, USA) regarding the management and use of laboratory animals, as stipulated in the license issued by the National Animal Experiment Board of Finland. 8-12 week old C57Bl / 6J mice were housed in a light-controlled environment (lights on from 7 a.m. to 8 p.m.) at a standard temperature (22 ± 1°C) and were given free access to food and water.

[0140] MPTP solution was prepared by dissolving MPTP hydrochloride in sterile saline at a concentration of 2.42 mg / mL. This corresponds to a concentration of 2.0 mg / mL of the active compound. To induce Parkinson's disease, MPTP was administered by intraperitoneal injection at a dose of 20 mg / kg twice daily. MPTP injections, or saline alone for control mice, were administered at 4-hour intervals over two consecutive days (day 0 and day 1).

[0141] ApoTf protein was administered at a concentration of 51.5 mg / mL in sterile PBS at pH 7.4. Mice were either administered ApoTf at a dose of 350 mg / kg by intraperitoneal injection, or control mice were administered PBS alone. ApoTf was administered once daily from days 1 to 7, with the first ApoTf treatment dose given 1 hour after the last MPTP dose on day 1.

[0142] Mice were subjected to kinematic gait analysis using the Motorater (TSE Systems GmbH, Bad Homburg, Germany) testing system on day 7. The animals were tested during their light cycle between 7 a.m. and 8 p.m. Prior to the motion and gait analysis session, 31 marks were placed on the mice's bodies to facilitate data analysis of the captured video. Motion was captured from three different directions (below and on both sides) using a high-speed camera (300 fps).

[0143] The images of each captured mouse were first converted to a software-readable format. To obtain raw data, body markers were tracked and associated in each of the three directions. Then, different gait patterns and movements were extracted using custom software developed by Charles River Discovery Research Service Finland. The gait pattern and movement analysis evaluated 100 different parameters, including, but not limited to, stride time, swing time between strides, velocity, step length, posture, and limb coordination. The data were analyzed using principal component analysis (PCA). The overall gait analysis is based on PCA of all parameters for each mouse, and the obtained values ​​represent the overall difference measured as "distance" between the control mouse and the MPTP mouse or MPTP plus ApoTf mouse. The control mouse (control) is set to a value of 0, and the "distance from control" is shown for MPTP-only mice (MPTP) or MPTP mice that have undergone ApoTf processing (MPTP→ApoTf). The values ​​are shown as mean + / - SEM (n=15).

[0144] array The sequences mentioned in the preceding paragraph are briefly described below in fasta format.

[0145] Sequence ID 1: Human transferrin [UniProt Q06AH7] protein sequence [ka]

[0146] Sequence ID 2: Human lactoferrin [UniProt P02788] protein sequence [ka]

[0147] Sequence ID 3: Y188F transferrin N-lobe mutant protein [ka]

[0148] Sequence ID 4: Y95F / Y188F transferrin N-lobe mutant protein [ka]

[0149] Sequence ID 5: Y426F / Y517F transferrin C-lobe mutant protein [ka]

[0150] Sequence ID 6: BDNF [ka]

[0151] Sequence ID 7: GDNF [ka]

[0152] Sequence ID 8: CNTF [ka]

[0153] Sequence ID 9: PACAP [ka]

Claims

1. A composition for use in a method for stimulating the development of new nerve cells through neurogenesis in patients suffering from neurodegenerative events, 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 protein 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 neurodegenerative event is caused by a neurodegenerative disease.

7. The composition according to any one of claims 1 to 6, wherein the neurodegenerative event is a neurodegenerative disease selected from the group consisting of Parkinson's disease, frontotemporal dementia, Alzheimer's disease, mild cognitive impairment, diffuse Lewy body disease, Lewy body dementia, demyelinating disease, amyotrophic lateral sclerosis, Huntington's disease, Creutzfeldt-Jakob disease, corticobasal ganglia ganglion degeneration, peripheral neuropathy, progressive supranuclear palsy, spinocerebellar degeneration, spinal ataxia, Friedreich's ataxia, cerebellar cortical degeneration, neurogenic muscular atrophy, anterior horn cell degeneration, infantile spinal muscular atrophy and juvenile spinal muscular atrophy, subacute sclerosing panencephalitis, Haller-Holden-Spats disease, boxer's dementia, Pick's disease, tauopathy, synuclein disease, and combinations thereof.

8. The composition according to any one of claims 1 to 7, wherein the method further comprises the step of administering a therapeutically effective amount of serum or plasma protein to the patient in addition to transferrin.

9. The composition according to claim 8, wherein serum or plasma protein and transferrin are administered as unital dosage forms.

10. The composition according to claim 8 or 9, wherein the serum or plasma protein is selected from the group consisting of albumin, alpha-1 antitrypsin / alpha-1 proteinase inhibitors, antithrombin, polyclonal immunoglobulins, polyspecific immunoglobulins, C1 esterase inhibitors, transthyretin, and combinations thereof.

11. The composition according to any one of claims 1 to 10, wherein the method further comprises the step of administering, in addition to transferrin, 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. 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 claim 11 or 12, selected from the group consisting of the following.

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

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 16, 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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