COMPOSITIONS HAVING NEUROREGENERATIVE APPLICATIONS

Abstract

Pharmaceutical compositions containing transferrin or lactoferrin for use in promoting or inducing the generation new neural cells in a patient that has suffered a neurodegenerative event. The neurodegenerative event may be caused by a neurodegenerative disease such as Alzheimer's, Parkinson's, Huntington's, or amyotrophic lateral sclerosis. Ideally, the transferrin and/or lactoferrin have a low iron saturation.

Claims

1. A method of promoting and or inducing generation of new neural cells in a patient that has suffered a neurodegenerative event, the method comprising administering a therapeutically effective amount of a protein selected from transferrin, lactoferrin, and combinations thereof to the patient in need thereof.

2. The method of claim 1, wherein the therapeutically effective amount of transferrin or lactoferrin administered to the patient has an iron saturation of less than about 20%.

3. The method of claim 1, wherein the protein is human transferrin.

4. The method of claim 1, wherein the transferrin is plasma-derived or recombinant.

5. The method of claim 4, wherein the recombinant transferrin is a mutant transferrin selected from the group consisting of: i) Y188F mutant comprising the amino acid sequence set forth in SEQ ID NO: 3; ii) Y95F/Y188F mutant comprising the amino acid sequence set forth in SEQ ID NO: 4; iii) Y426F/Y517F mutant comprising the amino acid sequence set forth in SEQ ID NO: 5; and iv) combinations thereof.

6. The method of claim 1, wherein the transferrin is a domain of a fusion protein, and the fusion partner is an immunoglobulin Fc domain.

7. The method of claim 1, wherein the neurodegenerative event is caused by a neurodegenerative disease.

8. The method of claim 1, 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, Dementia with Lewy bodies type, demyelinating diseases such as multiple sclerosis and acute transverse myelitis, amyotrophic lateral sclerosis, Huntington's disease, Creutzfeldt-Jakob disease, corticobasal ganglionic degeneration, peripheral neuropathy, progressive supranuclear Palsy, spinocerebellar degenerations, spinal ataxia, Friedreich's ataxia, cerebellar cortical degenerations, neurogenic muscular atrophies, anterior horn cell degeneration, infantile spinal muscular atrophy, and juvenile spinal muscular atrophy, subacute sclerosing panencephalitis, Hallervorden-Spatz disease, dementia pugilistica, Pick's disease, tauopathies, synucleinopathies, and combinations thereof.

9-21. (canceled)

22. A method of stimulating neural cell development in a patient that has suffered a neurodegenerative event, the method comprising administering a therapeutically effective amount of a protein selected from transferrin, lactoferrin, and combinations thereof to the patient in need thereof.

23. The method of claim 22, wherein the therapeutically effective amount of transferrin or lactoferrin administered to the patient has an iron saturation of less than about 20%.

24. The method of claim 22, wherein the protein is human transferrin.

25. The method of claim 22, wherein the transferrin is plasma derived, or recombinant.

26. The method of claim 25, wherein the recombinant transferrin is a mutant transferrin selected from the group consisting of: i) Y188F mutant comprising the amino acid sequence set forth in SEQ ID NO: 3; ii) Y95F/Y188F mutant comprising the amino acid sequence set forth in SEQ ID NO: 4; iii) Y426F/Y517F mutant comprising the amino acid sequence set forth in SEQ ID NO: 5; and iv) combinations thereof.

27. The method of claim 22, wherein the transferrin is a domain of a fusion protein, and the fusion partner is an immunoglobulin Fc domain.

28. The method of claim 22, wherein the neurodegenerative event is caused by a neurodegenerative disease.

29-42. (canceled)

43. A stable pharmaceutical composition comprising: a therapeutically effective amount of a protein selected from transferrin, lactoferrin, and combinations thereof, and at least one pharmaceutically acceptable excipient, wherein the therapeutically effective amount of a protein selected from transferrin, lactoferrin, and combinations thereof has an iron saturation of less than about 25%.

44. The pharmaceutical composition of claim 43, wherein the protein is human transferrin.

45. The pharmaceutical composition of claim 43, wherein the transferrin is plasma derived, or recombinant.

46. The pharmaceutical composition of claim 45, wherein the recombinant transferrin is a mutant transferrin selected from the group consisting of: Y188F mutant comprising the amino acid sequence set forth in SEQ ID NO: 3; Y95F/Y188F mutant comprising the amino acid sequence set forth in SEQ ID NO: 4; Y426F/Y517F mutant comprising the amino acid sequence set forth in SEQ ID NO: 5; and combinations thereof.

47. The pharmaceutical composition of claim 43, wherein the transferrin is a domain of a fusion protein, and the fusion partner is an immunoglobulin Fc domain.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0110] Additional features and advantages of the present invention will be made clearer in the appended drawings, in which:

[0111] FIGS. 1A to 1D show the induction of neurite outgrowth, and proliferation in SH-SYSY cells and increased β-III-tubulin protein concentrations in response to apo-transferrin;

[0112] FIGS. 2A to 2B demonstrate that apo-transferrin induces primary human neural progenitor cells to become β-III-tubulin protein positive neurons and GFAP protein positive astrocytes cells;

[0113] FIG. 3A plots the effect of deferoxamine mesylate at various concentrations relative to apo-transferrin on neurite outgrowth in SH-SYSY cells;

[0114] FIG. 3B illustrates the efficacy of a transferrin mutant having reduced iron binding capacity on promoting neurite outgrowth in SH-SYSY cells;

[0115] FIG. 4 plots the effect of various different proteins on neurite outgrowth in SH-SYSY cells;

[0116] FIG. 5 plots the effect of IOX2, a prolyl hydroxylase inhibitor, on neurite outgrowth in SH-SYSY cells;

[0117] FIGS. 6A & 6B illustrate the role of iron saturation on the efficacy of transferrin in promoting neurite outgrowth in SH-SY5Y cells;

[0118] FIGS. 7A to 7D plots the effect of apo-transferrin in combination with other neurotrophic proteins/peptide fragments on neurite outgrowth in SH-SY5Y cells;

[0119] FIG. 8 plots the effect of apo-transferrin in combination with the small molecule Y-27632 on neurite outgrowth in SH-SY5Y cells; and

[0120] FIG. 9 demonstrates the positive regenerative effect of apo-transferrin in MPTP-induced Parkinson's Disease in mice.

DETAILED EXAMPLES OF THE INVENTION

[0121] It should be readily apparent to one of ordinary skill in the art that the examples disclosed herein below represent generalised examples only, and that other arrangements and methods capable of reproducing the invention are possible and are embraced by the present invention.

Example 1: Apo-Transferrin (ApoTf) Induces Differentiation and Neurite Outgrowth in SH-SY5Y Cells in a Dose Responsive Manner

[0122] Transferrin is utilized in cell culture and in-vivo to deliver iron as a nutrient to cells. This is typically accomplished through the actions of holo-transferrin (HoloTf) binding to, and endocytosis by, its cognate receptor CD71, the transferrin receptor 1 (TfR1). Transferrin is typically believed to provide cells with iron as a means to promote and sustain metabolic activity. The present inventors have surprisingly found that apo-transferrin, the iron-free form of transferrin protein, induces differentiation of a very common research model of neurons, SH-SY5Y cells. Induction of neuronal differentiation was assessed by morphological parameters of neurite formation (a key 30 element typically used as a marker of neuronal differentiation, neuronal health, and function) according to the procedures of Agholme, 2010. J. of Alzheimer's Disease. Vol. 20:1p 069-108; and Dyberg et al., 2017. PNAS Vol 114 (32), E6603-E6612.

[0123] Undifferentiated SH-SY5Y cells were seeded into 96 well clear bottom plates in media containing 0.1% FBS. A serum-free base media was utilized as recommended by the supplier for SH-SY5Y cells (Sigma, Cat #94030304-1VL). Twenty-four hours after seeding cells, a 3× stock solution of ApoTf, final concentrations indicated on the x-axis, in serum free base media was added to the cells. ApoTf was obtained and purified from pooled human plasma and dosed at a final concentration of 0.2 mg/mL. Cells were allowed to differentiate for 6 days. Neurite growth was assessed by imaging and image analysis. At the time of analysis, a 10× solution of Tubulin Tracker (Molecular Probes, T34075) and Hoechst 33342 (Molecular Probes #H3570) nuclear stain was prepared.

[0124] Briefly, Tubulin Tracker dissolved in DMSO was diluted 1:1 with Pluronic F-127 and further diluted into HBSS to generate a 10× solution. Hoechst 33342 was added to the HBSS-Tubulin Tracker solution at 10 μg/mL to generate a 10× nuclear stain. The 10× staining solution (10 μL) was added directly to treated assay wells and incubated at 37° C. for 30 minutes. Following incubation, 110 μL of 0.4% Trypan Blue was added directly to assay wells and imaged on a Molecular Devices Nano imaging instrument. Nine images/well were acquired in the blue (Nuclei) and green (Tubulin) fluorescent channels for each image.

[0125] After obtaining images, the MetaExpress Neurite Outgrowth analysis module (Molecular Devices) was used to identify cells, cell bodies, and quantify neurites. The total number of neurite branches were divided by the total number of cells imaged to account for different numbers of cells in each test well. The Outgrowth Fold Change was determined by setting the untreated control cells to a value of 1 with all other treatments shown relative to untreated control.

[0126] From FIG. 1A it is evident that apo-transferrin was able to induce neurite outgrowth in a dose dependent manner. Incremental increases in apo-transferrin concentration, up to a maximum of 0.8 mg/mL, were associated with an improved outgrowth response in the SH-SY5Y cells. This phenomenon is counterintuitive to the known function of transferrin, which primarily acts in the holo- or iron-laden form of transferrin.

[0127] FIG. 1B illustrates that apo-transferrin induces a concentration dependent increase in cell numbers. Increased cell number is indicative of increased cell proliferation, up to the maximum tested dose of 0.8 mg/mL apo-transferrin.

[0128] FIG. 1C provides visual comparison of SH-SY5Y cells treated with 0.1 mg/mL ApoTf (lower panels) to an untreated control (upper panels). Left panels show nuclear staining with Hoechst 33342. Right images show tubulin staining of cell bodies and neurites. From FIG. 1C it is apparent that ApoTf had a profound effect on promoting cell proliferation, and subsequently/simultaneously promoting induction of neurite/tubulin outgrowth.

[0129] Additionally, as shown in FIG. 1D, it was found that apo-transferrin treatment caused an increase in β-III-tubulin protein, a well-characterized, traditional marker of neurons. In this experiment, the SH-SY5Y cells were differentiated as described supra. At the time of analysis, cells were fixed with paraformaldehyde, stained for β-III-tubulin (R&D Systems, MAB1195), and imaged on a Molecular Devices Nano imaging instrument. Image analysis was performed by assessing the fluorescent intensity of cells stained with β-III-tubulin. Background from secondary antibody alone was subtracted from all values. Values are shown with standard deviations as “β-III-Tubulin Staining Intensity” for the indicated conditions.

[0130] SH-SY5Y Cells

[0131] By “SH-SY5Y cells” the present specification means a subcloned cell line derived from the SK-N-SH neuroblastoma cell line. It serves as a model for neurodegenerative disorders since the cells can be converted to various types of functional neural cells by the addition of specific compounds. In addition, the SH-SY5Y cell line has been used widely in experimental neurological studies, including analysis of neuronal differentiation, metabolism, and function related to neurodegenerative processes, neurotoxicity, and neuroprotection.

[0132] Outlined herein under are peer reviewed citations referencing the SH-SY5Y cell line as a predictive model for various neurodegenerative disorders. The list does not constitute an admission of prior art by the inventors, rather it serves to illustrate the skilled person's knowledge of the utility of the SH-SY5Y cell line as a predictive model for neurodegenerative disorders.

[0133] Neurogenesis [0134] 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, 934-943. [0135] Fagerstrom et al. Protein Kinase C-epsilon Implicated in Neurite Outgrowth in Differentiating Human Neuroblastoma Cells. Cell Growth & Differentiation Vol. 7, 775-785, June 1996.

[0136] Mood Stabilization (Depression) [0137] Yuan et al. The Mood Stabilizer Valproic Acid Activates Mitogen-activated Protein Kinases and Promotes Neurite Growth. JBC Vol. 276, No. 34, Issue of August 24, pp. 31674-31683, 2001. [0138] Tatro et al. Modulation of Glucocorticoid Receptor Nuclear Translocation in Neurons by Immunophilins FKBP51 and FKBP52: Implications for Major Depressive Disorder. Brain Res. 2009 Aug. 25; 1286: 1-12. [0139] 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, 1054-1064. [0140] 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 Issue 1, p 49-62.

[0141] Tauopathy (Alzheimer's Disease, FTD, and Other Neurodegenerative Diseases with Abnormal Tau) [0142] 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) 993-1000. [0143] Seidel et. al. Induced Tauopathy in a Novel 3D-Culture Model Mediates Neurodegenerative Processes: A Real-Time Study on Biochips. PLOS One. (November 2012) Volume 7 Issue 11. e49150. [0144] Karch et al. Extracellular Tau Levels Are Influenced by Variability in Tau That Is Associated with Tauopathies. JBC VOL. 287, NO. 51, pp. 42751-42762, Dec. 14, 2012.

[0145] Alzheimer's Disease [0146] Pettifer et al. Guanosine protects SH-SY5Ycells against b-amyloid-induced apoptosis. NeuroReport 2004 15(5):833-836. [0147] Tanii et al. Alzheimer's Disease Presenilin-1 Exon 9 Deletion And L250s Mutations Sensitize SH-SYSY Neuroblastoma Cells To Hyperosmotic Stress-Induced Apoptosis. Neuroscience Vol. 95, No. 2, pp. 593-601, 2000. [0148] Li et al. Beta-amyloid induces apoptosis in human-derived neurotypic SH-SYSY cells. Brain Res. 1996 Nov. 4; 738(2):196-204.

[0149] ALS and Frontotemporal Dementia [0150] Lee et al. Hexanucleotide Repeats in ALS/FTD Form Length-Dependent RNA Foci, Sequester RNA Binding Proteins, and Are Neurotoxic. Cell Reports 5, 1178-1186, Dec. 12, 2013. [0151] Farg et al. C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking. Human Molecular Genetics, 2014, Vol. 23, No. 13. [0152] Nonaka et al. Phosphorylated and ubiquitinated TDP-43 pathological inclusions in ALS and FTLD-U are recapitulated in SH-SYSY cells. FEBS Letters 583 (2009) 394-400.

[0153] Parkinson's Disease [0154] Xing et al. Protective effects and mechanisms of Ndfipl on SH-SYSY cell apoptosis in an in vitro Parkinson's disease model. Genetics and Molecular Research 15 (2): gmr.15026963. [0155] Jung et al. Rosiglitazone protects human neuroblastoma SH-SYSY cells against MPP+ induced cytotoxicity via inhibition of mitochondrial dysfunction and ROS production. Journal of the Neurological Sciences 253 (2007) 53-60. [0156] Choi et al. Signaling Pathway Analysis of MPP+-treated Human Neuroblastoma SH-SYSY Cells. Biotechnology and Bioprocess Engineering 19: 332-340 (2014).

[0157] Friedreich's Ataxia [0158] 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 2807-2822.

[0159] Huntington's Disease [0160] Banez-Coronel et al. A Pathogenic Mechanism in Huntington's Disease Involves Small CAG-Repeated RNAs with Neurotoxic Activity. Neuroscience Research Volume 53, Issue 3, November 2005, Pages 241-249. [0161] Vidoni et al. Resveratrol protects neuronal-like cells expressing mutant Huntingtin from dopamine toxicity by rescuing ATG4-mediated autophagosome formation. Neurochemistry International 117 (2018) 174-187. [0162] Vidoni et al. Dopamine exacerbates mutant Huntingtin toxicity via oxidative mediated inhibition of autophagy in SH-SYSY neuroblastoma cells: Beneficial effects of anti-oxidant therapeutics. Neurochemistry International 101 (2016) 132-143. [0163] 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) 271-281.

Example 2: The Effect of ApoTf on β-III-Tubulin and GFAP Protein Concentrations in Primary Human Neural Progenitor Cells

[0164] The neurogenic effects of ApoTf also translate to primary human brain 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 FIGS. 2A and 2B, apo-transferrin dramatically increases the percentage of cells differentiated to neurons (% β-III-tubulin positive cells, 2A) and astrocytes (% GFAP positive cells, 2B), relative to cells without apo-transferrin, from a culture of primary human brain-derived neural progenitor cells.

[0165] Neural progenitor cells maintained as neurospheres were obtained from Lonza (PT-2599). Cells were thawed from a frozen vial of neurospheres and cultured in Human NeuroCult™ NS-A Complete Proliferation media (Stemcell Technologies) for 2 weeks. Neurospheres were dissociated to single cells and plated in Laminin coated wells of assay plates. The neural progenitor cells were seeded in NeuroCult™ NS-A Basal media containing 1/10th concentration of the recommended proliferation supplements, in the absence or presence of ApoTf (0.8 mg/mL) for 72 hours. At the time of analysis, cells were fixed with paraformaldehyde, stained for β-III-tubulin (R&D Systems, MAB1195) and GFAP (Invitrogen, PA3-16727), and imaged on a Molecular Devices Nano imaging instrument. Image analysis was performed by assessing the relative numbers of cells staining positive for β-III-tubulin or GFAP. Values for the indicated conditions are shown with standard deviations as “% β-III-Tubulin Positive” cells (FIG. 2A) or “% GFAP Positive” cells (FIG. 2B).

Example 3: Iron Chelation is not the Sole Mode of Action for Neurogenesis by ApoTf

[0166] Deferoxamine mesylate (DFO) is a small molecule iron chelator utilized in clinical practice for iron overload. Like ApoTf, DFO has high affinity binding constants for iron; although only a single iron binding site. The effect of DFO on neurite outgrowth was investigated. ApoTf was tested at a concentration near the bottom of its functional dose curve and compared to DFO's ability to induce neurite outgrowth. ApoTf tested at 2.4 μM (0.2 mg/mL) has two iron binding sites and therefore is comparable to the single iron binding site of DFO at 4.8 μM.

[0167] Undifferentiated SH-SYSY cells were seeded and treated as described in Example 1. Neurite growth was assessed by imaging and image analysis. At the time of analysis, a 10× solution of Tubulin Tracker (Molecular Probes, T34075) and Hoechst 33342 (Molecular Probes #H3570) nuclear stain was prepared. Briefly, Tubulin Tracker dissolved in DMSO was diluted 1:1 with Pluronic F-127 and further diluted into HBSS to generate a 10× solution. Hoechst 33342 was added to the HBSS-Tubulin Tracker solution at 10 μg/mL to generate at 10× nuclear stain. The 10× staining solution (10 μL) was added directly to treated assay wells and incubated at 37° C. for 30 minutes. Following incubation, 110 μL of 0.4% Trypan Blue was added directly to assay wells and imaged on a Molecular Devices Nano imaging instrument. Nine images/well were acquired in the blue (Nuclei) and green (Tubulin) fluorescent channels for each image. After obtaining images, the MetaExpress Neurite Outgrowth analysis module (Molecular Devices) was used to identify cell bodies and quantify neurites. The total number of neurite branches were divided by the total number of cells imaged to account for different numbers of cells. The Outgrowth Fold Change was determined by setting the untreated control to a value of 1 with all other treatments shown relative to untreated control. ApoTf was obtained and purified from pooled human plasma and dosed at a final concentration of 0.2 mg/mL. Deferoxamine mesylate (DFO) was obtained from Tocris (Cat #5764), resuspended and stored by the manufacturer's recommendations. Concentrations of DFO that were assessed for neurogenic properties are indicated on the x-axis.

[0168] From FIG. 3A it can been seen that DFO shows maximal neurite outgrowth between 1-3 μM, with little neurite formation beyond that concentration, whereas ApoTf continues to increase differentiation even up to 9.9 μM (0.8 mg/mL; 20 μM iron binding sites). These data suggest that while iron chelation may play a role in neurite outgrowth, it is not the primary mechanism-of-action; another unidentified functional aspect of ApoTf must also play a role in its neurogenic ability.

[0169] The present inventors further sought to determine whether a reduction of transferrin's iron-binding activity by mutation of the N-terminal iron-binding site was sufficient to mediate neurogenesis.

[0170] Undifferentiated SH-SYSY cells were treated as described in Example 1. Neurite growth was assessed by imaging and image analysis. At the time of analysis, a 10× solution of Tubulin Tracker (Molecular Probes, T34075) and Hoechst 33342 (Molecular Probes #H3570) nuclear stain was prepared. Briefly, Tubulin Tracker dissolved in DMSO was diluted 1:1 with Pluronic F-127 and further diluted into HBSS to generate a 10× solution. Hoechst 33342 was added to the HBSS-Tubulin Tracker solution at 10 μg/mL to generate at 10× nuclear stain. The 10× staining solution (10 μL) was added directly to treated assay wells and incubated at 37° C. for 30 minutes. Following incubation, 110 μL of 0.4% Trypan Blue was added directly to assay wells and imaged on a Molecular Devices Nano imaging instrument. Nine images/well were acquired in the blue (Nuclei) and green (Tubulin) fluorescent channels for each image. After obtaining images, the MetaExpress Neurite Outgrowth analysis module (Molecular Devices) was used to identify cell bodies and quantify neurites. The total number of neurite branches were divided by the total number of cells imaged to account for different numbers of cells. The Outgrowth Fold Change was determined by setting the untreated control to a value of 1 with all other treatments shown relative to untreated control. All proteins were dosed at a final concentration of 0.2 mg/mL.

[0171] 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 the N-lobe mutant Tf (N-mut rec ApoTf; SEQ ID NO: 4) were obtained by cell culture expression from 293-6E cells.

[0172] Briefly, wild-type human transferrin (SEQ ID NO:1) and N-lobe mutant human transferrin (SEQ ID 4) sequences were cloned into mammalian expression plasmids containing N-terminal 6×HIS tag and TEV cleavage sites. The expression plasmids were transfected into the 293-6E cell line, with subsequent harvest of proteins from the cell culture supernatant. Proteins were purified on NI-NTA columns and eluted after washing. TurboTEV protease was used to cleave the N-terminal 6×HIS tag and additional amino acids from the transferrin proteins. Following TEV cleavage, the transferrin proteins were separated from cleaved 6×HIS tag and uncleaved protein by a second Ni-NTA capture column. The flow-through fraction of Ni-NTA capture column was then subject to low pH treatment to remove any potential residual iron bound to these proteins, buffer exchanged to PBS pH 7.4, concentrated, and sterile filtered for final use.

[0173] From FIG. 3B we see that plasma-derived human serum albumin (pdHSA) did not affect neurogenesis. However, both ApoTf and recombinant ApoTf did induce neurogenesis of SH-SYSY. The ApoTf mutant (N-mut rec ApoTf) with reduced iron-binding capacity was almost equal to that of ApoTf and rec ApoTf at inducing differentiation of the SH-SYSY cells. Iron-binding does not appear to be the sole mechanism of action for the neurogenic potential of ApoTf.

Example 4: Neurogenic Effects on SH-SY5Y are Specific to Apo-Transferrin and Apo-Lactoferrin

[0174] As the role of iron chelation in ApoTf's neurogenic ability was found to unclear from Example 3 the present inventors determined whether other iron binding proteins can also mediate neurogenesis of SH-SYSY cells.

[0175] Undifferentiated SH-SYSY cells were treated as described in Example 1. Neurite growth was assessed by imaging and image analysis. At the time of analysis, a 10× solution of Tubulin Tracker (Molecular Probes, T34075) and Hoechst 33342 dissolved in DMSO was diluted 1:1 with Pluronic F-127 and further diluted into HBSS to generate a 10× solution. Hoechst 33342 was added to the HBSS-Tubulin Tracker solution at 10 μg/mL to generate at 10× nuclear stain. The 10× staining solution (10 μL) was added directly to treated assay wells and incubated at 37° C. for 30 minutes. Following incubation, 110 μL of 0.4% Trypan Blue was added directly to assay wells and imaged on a Molecular Devices Nano imaging instrument. Nine images/well were acquired in the blue (Nuclei) and green (Tubulin) fluorescent channels for each image. After obtaining images, the MetaExpress Neurite Outgrowth analysis module (Molecular Devices) was used to identify cell bodies and quantify neurites. The total number of neurite branches were divided by the total number of cells imaged to account for different numbers of cells. The Outgrowth Fold Change was determined by setting the untreated control to a value of 1 with all other treatments shown relative to 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 (equine) was obtained from Sigma; apo-lactoferrin was obtained from Athens Research & Technology. All proteins were dosed at a final concentration of 0.2 mg/mL.

[0176] From FIG. 4 we see that neither bovine serum albumin (BSA) nor a low-affinity iron binding form of human serum albumin affected neurogenesis. For further information on the low-affinity iron binding form of human serum albumin (rHSA) see Silva et al., 2009. Biochimica et Biophysica Acta, Vol 1794, p 1449-1458. Holo-transferrin (HoloTf), the iron-saturated form of transferrin, was also unable to induce differentiation of the SH-SYSY cells.

[0177] Surprisingly, apo-ferritin, the iron-poor form of ferritin, another high-affinity iron binding protein with multiple iron binding sites, was ineffective at inducing differentiation of the SH-SYSY cells. This furthered the hypothesis that iron binding is the sole mechanism of action for the neurogenic potential of ApoTf. Unexpectedly, apo-lactoferrin also induced differentiation of these cells. Apo-lactoferrin is a structural and functional homologue of apo-transferrin but found in breast milk rather than plasma.

[0178] Apo-lactoferrin has 61% identity with apo-transferrin, whereas apo-ferritin and Human Serum Albumin (HSA) are structurally unrelated to either apo-transferrin or apo-lactoferrin.

Example 5: ApoTf Induced Differentiation of SH-SY5Y Cells is not Through Hypoxia Inducible Factor 1α (HIF-1α)

[0179] It has been reported that both ApoTf and HoloTf can induce HIF-1α production leading to associated neuroprotective effects (US2016008437 to Grifols Worldwide Operations limited, the contents of which are incorporated herein by reference). While this is a beneficial attribute prior to death of a neuron, neuroprotection does not benefit the patient once a neuronal cell is dead. Neurogenesis, on the other-hand, benefits the patient after the insult because it can regenerate new neuronal cells.

[0180] In substantiation of the premises that ApoTf is mediating neurogenesis outside of the HIF pathway the present inventors tested a well-known, highly specific prolyl hydroxylase (PHD2) inhibitor in the SH-SY5Y cell differentiation assay. 10 λ2 (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 actions on PHD2. See Chowdhury et al., 2013. ACS Chem. Biol. Vol 8, p 1488. IOX2 has an 1050 of 22 nM for inhibition of PHD2 and can induce up-regulation of HIF-1α in undifferentiated SH-SY5Y with concentrations as little as 1 μM (Ross, US2016008437 supra).

[0181] Undifferentiated SH-SY5Y cells were seeded and treated as described in Example 1. Neurite growth was assessed by imaging and image analysis. At the time of analysis, a 10× solution of Tubulin Tracker (Molecular Probes, T34075) and Hoechst 33342 (Molecular Probes #H3570) nuclear stain was prepared. Briefly, Tubulin Tracker dissolved in DMSO was diluted 1:1 with Pluronic F-127 and further diluted into HBSS to generate a 10× solution. Hoechst 33342 was added to the HBSS-Tubulin Tracker solution at 10 μg/mL to generate at 10× nuclear stain. The 10× staining solution (10 μL) was added directly to treated assay wells and incubated at 37° C. for 30 minutes. Following incubation, 110 μL of 0.4% Trypan Blue was added directly to assay wells and imaged on a Molecular Devices Nano imaging instrument. Nine images/well were acquired in the blue (Nuclei) and green (Tubulin) fluorescent channels for each image. After obtaining images, the MetaExpress Neurite Outgrowth analysis module (Molecular Devices) was used to identify cell bodies and quantify neurites. The total number of neurite branches were divided by the total number of cells imaged to account for different numbers of cells. The Outgrowth Fold Change was determined by setting the untreated control to a value of 1 with all other treatments shown relative to untreated control. ApoTf was obtained and purified from pooled human plasma and dosed at a final concentration of 0.2 mg/mL. IOX2 was obtained from Tocris (Cat #4451), resuspended and stored by the manufacturer's recommendations.

[0182] From FIG. 5 it is evident that no neurite outgrowth or differentiation was observed in the IOX2-treated cells. Even at very high concentrations of 4 μM IOX2 no effect was observable (4-fold higher than concentrations reported in US2016008437 to induce of HIF-1α in SH-SY5Y, and over 180-fold higher than the concentration that Chowdhury determined as the IC.sub.50 for PHD2 proteins). These data, in combination with the lack of neurogenesis with HoloTf (Example 4), indicate that HIF-1α does not play a role in differentiating SH-SY5Y cells.

Example 6: Role of Iron Saturation in Transferrin Efficacy

[0183] ApoTf, with various purities and iron saturation amounts, as outlined in Table 1 were assessed for their neurogenic potential. The transferrin samples were prepared according to the procedures/methodology known by those skilled in the art and detailed in section 21.4 of L von Bonsdorff, et al., Transferrin, Ch 21, pg 301-310, Production of Plasma Proteins for Therapeutic Use, Eds. J. Bertolini, et al., Wiley, 2013 [Print ISBN:9780470924310 |Online ISBN:9781118356807], the contents of which are incorporated herein by reference.

[0184] Protein purity was determined by SDS-PAGE. Iron saturation levels were determined using ICP-AES in accordance with the procedures outlined in Manley et al., J Biol Inorg Chem (2009) 14:61-74, the contents of which are incorporated herein by reference.

TABLE-US-00001 TABLE 1 Protein Iron Saturation Sample Name Purity (%) (%) Source ApoTransferrin A 99.11 0.27 Grifols - prepared in house ApoTransferrin B 98.57 0.59 Grifols - prepared in house ApoTransferrin C 96.72 0.24 Grifols - prepared in house ApoTransferrin D 94.35 Not Athens Research & Determined Technology Inc., Cat# 16-16A32001-BPG HoloTransferrin 99.0 100 Grifols - prepared in house

[0185] Undifferentiated SH-SY5Y cells were treated as described in Example 1. Neurite growth was assessed by imaging and image analysis. At the time of analysis, a 10× solution of Tubulin Tracker (Molecular Probes, T34075) and Hoechst 33342 (Molecular Probes #H3570) nuclear stain was prepared. Briefly, Tubulin Tracker dissolved in DMSO was diluted 1:1 with Pluronic F-127 and further diluted into HBSS to generate a 10× solution. Hoechst 33342 was added to the HBSS-Tubulin Tracker solution at 10 μg/mL to generate at 10× nuclear stain. The 10× staining solution (10 μL) was added directly to treated assay wells and incubated at 37° C. for 30 minutes. Following incubation, 110 μL of 0.4% Trypan Blue was added directly to assay wells and imaged on a Molecular Devices Nano imaging instrument. Nine images/well were acquired in the blue (Nuclei) and green (Tubulin) fluorescent channels for each image. After obtaining images, the MetaExpress Neurite Outgrowth analysis module (Molecular Devices) was used to identify cell bodies and quantify neurites. The total number of neurite branches were divided by the total number of cells imaged to account for different numbers of cells. The Outgrowth Fold Change was determined by setting the untreated control to a value of 1 with all other treatments shown relative to untreated control.

[0186] FIG. 6A plots the effect of ApoTf A-D, purity & iron content outlined in Table 1, dosed at a final concentration of 0.2 mg/mL on neurite outgrowth in SH-SY5Y cells. FIG. 6B plots transferrin with various iron saturation levels (listed on the X-axis) dosed at final concentrations of 0.2 mg/mL on neurite outgrowth in SH-SY5Y cells.

[0187] ApoTf (<0.3% Saturation) and, HoloTf (100% Saturation) were prepared after purification of transferrin from pooled human plasma as outlined in von Bonsdorff, vide supra. The various iron saturation contents were generated by mixing ApoTf and HoloTf to generate the indicated percent saturations plotted in FIG. 6B.

[0188] From FIG. 6A we see that all ApoTf preparations (ApoTf A-D), even the sample with a protein purity of only 94%, were able to induce neurogenic differentiation of SH-SY5Y. FIG. 6B illustrates effect the degree of iron saturation had on the ability of transferrin to induce differentiation of the SH-SY5Y cells. In this example, ApoTf or HoloTf with protein purities of at least 99% were mixed in various ratios to determine the effect of iron saturation/content. Transferrin with an iron saturation content less than 30% showed neurogenic potential.

Example 7: Apo-Transferrin Acts Synergistically with Neurotrophic Protein and Peptide Factors to Induce Differentiation

[0189] Several neurotrophic protein factors have been considered for clinical use for stimulation of neurogenesis in neurodegenerative conditions 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.

[0190] Proteins from three neurotrophic superfamilies were tested for function in combination with ApoTf. These neurotrophic proteins are: BDNF (brain-derived neurotrophic factor; NGF superfamily), GNDF (glial cell line-derived neurotrophic factor; TGF-β superfamily), and CNTF (cilliary neurotrophic factor-1; neurokine superfamily). In addition, another known neurotrophic peptide, PACAP (amino acids 1-38 of pituitary adenylate cyclase-activating polypeptide), was assessed for function in combination with ApoTf.

[0191] Undifferentiated SH-SY5Y cells were treated as described in Example 1. Neurite growth was assessed by imaging and image analysis. At the time of analysis, a 10× solution of Tubulin Tracker (Molecular Probes, T34075) and Hoechst 33342 dissolved in DMSO was diluted 1:1 with Pluronic F-127 and further diluted into HBSS to generate a 10× solution. Hoechst 33342 was added to the HBSS-Tubulin Tracker solution at 10 μg/mL to generate at 10× nuclear stain. The 10× staining solution (10 μL) was added directly to treated assay wells and incubated at 37° C. for 30 minutes. Following incubation, 110 μL of 0.4% Trypan Blue was added directly to assay wells and imaged on a Molecular Devices Nano imaging instrument. Nine images/well were acquired in the blue (Nuclei) and green (Tubulin) fluorescent channels for each image. After obtaining images, the MetaExpress Neurite Outgrowth analysis module (Molecular Devices) was used to identify cell bodies and quantify neurites. The total number of neurite branches were divided by the total number of cells imaged to account for different numbers of cells. The Outgrowth Fold Change was determined by setting the untreated control to a value of 1 with all other treatments shown relative to untreated control.

[0192] In FIGS. 7A-7D ApoTf was dosed at a final concentration of 0.1 mg/mL either alone or in combination with the indicated neurotrophic factor. (A) BDNF was obtained from Peprotech (Cat #450-02) and dosed at 25 ng/mL. (B) GDNF was obtained from Peprotech (Cat #450-10) and dosed at 1000 ng/mL. (C) CNTF was obtained from Peprotech (Cat #450-13) and dosed at 250 ng/mL. (D) PACAP was obtained from Tocris (Cat #1186) and dosed at 200 nM. The abbreviation SF denotes serum free media.

[0193] Reviewing each of FIGS. 7A-7D it is apparent that each of the neurotrophic factors, and the peptide fragment induced differentiation of SH-SYSY cells to different degrees. In some cases, like BDNF, differentiation was not induced by the neurotrophic factor in the absence of ApoTf at the concentrations tested. In the all of the experiments presented, the neurotrophic factors combined with ApoTf induced greater differentiation than the molecules tested alone. Unexpectedly, ApoTf exhibits a synergistic effect with other neurotrophic factors and peptides on neurite outgrowth in SH-SYSY cells.

Example 8: Apo-Transferrin Acts Synergistically to Induce Differentiation with Neurogenic Small Molecules

[0194] The ability of ApoTf to act alongside non-protein based, neurogenic small molecule compounds was tested in Example 7. ApoTf was assessed in combination with the neurogenic 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 2 by small molecules has the known ability to induce neuronal differentiation, including SH-SY5Y cells. See Dyberg et al., 2017. PNAS Vol 114 (32), E6603-E6612.

[0195] Undifferentiated SH-SY5Y cells were treated as described in Example 1. Neurite growth was assessed by imaging and image analysis. At the time of analysis, a 10× solution of Tubulin Tracker (Molecular Probes, T34075) and Hoechst 33342 (Molecular Probes #H3570) nuclear stain was prepared. Briefly, Tubulin Tracker dissolved in DMSO was diluted 1:1 with Pluronic F-127 and further diluted into HBSS to generate a 10× solution. Hoechst 33342 was added to the HBSS-Tubulin Tracker solution at 10 μg/mL to generate at 10× nuclear stain. The 10× staining solution (10 μL) was added directly to treated assay wells and incubated at 37° C. for 30 minutes. Following incubation, 110 μL of 0.4% Trypan Blue was added directly to assay wells and imaged on a Molecular Devices Nano imaging instrument. Nine images/well were acquired in the blue (Nuclei) and green (Tubulin) fluorescent channels for each image. After obtaining images, the MetaExpress Neurite Outgrowth analysis module (Molecular Devices) was used to identify cell bodies and quantify neurites. The total number of neurite branches were divided by the total number of cells imaged to account for different numbers of cells. The Outgrowth Fold Change was determined by setting the untreated control to a value of 1 with all other treatments shown relative to untreated control. ApoTf was dosed 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 (Cat #1254) and dosed at 50 μM.

[0196] FIG. 8 illustrates that Y-27632 itself is a strongly neurogenic compound, however, in the presence of ApoTf, the neurogenic effect was synergistic showing an effect beyond that exhibited by either molecule alone. The ability of ApoTf to act synergistically with a number of known protein, peptide, and small molecule neurogenic entities is an unexpected and surprising finding.

Example 9: Improved Gait and Movement by Apo-Transferrin Treatment in a Mouse Model of Parkinson's Disease

[0197] To illustrate that the above in-vitro results would successfully translate into positive clinical effects the inventors trialled the 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 and induce Parkinson's disease in the mice. For more detail see Sedelis et al., Behavioural Brain Research 125 (2001), 109-122; Przedborski and Vila, Clinical Neuroscience Research 1 (2001), 407-418.

[0198] Destruction of dopaminergic neurons deleteriously effects animal movement. The movement and gait of the mice can be measured by video analysis. As shown in FIG. 9, a substantial alteration in movement and gait was observable in mice exposed to MPTP relative to control mice (n=15). Consistent with the findings in Examples 1-8, MPTP induced Parkinson's Disease in mice was significantly ameliorated by the administration of ApoTf (n=15). FIG. 9 demonstrates the neuroregenerative properties of ApoTf, in that ApoTf greatly improved movement dysfunction in the diseased mice, effectively normalizing the mice to the control animals.

[0199] Animal experiments were performed at Charles River Laboratories (Finland), as specified in the license authorized by the national Animal Experiment Board of Finland and according to the National Institutes of Health (Bethesda, Md., USA) guidelines for the care and use of laboratory animals. Eight to twelve-week-old, C57BI/6J mice were housed at a standard temperature (22±1° C.) and in a light-controlled environment (lights on from 7 am to 8 pm) with ad libitum access to food and water.

[0200] Solutions of MPTP were prepared by dissolving MPTP hydrochloride in sterile saline at 2.42 mg/mL; corresponding to 2.0 mg/mL of active compound. To induce Parkinson's Disease, the MPTP was given by intraperitoneal injection twice a day at 20 mg/kg. MPTP injections, or saline alone for control mice, were given at 4-hour intervals on two consecutive days (days 0 and 1).

[0201] ApoTf protein was administered in sterile PBS, pH 7.4 at a concentration of 51.5 mg/mL. The mice were dosed by intraperitoneal injection with ApoTf at 350 mg/kg or PBS alone for control mice. ApoTf was given once a day on days 1 through 7, with the first ApoTf treatment dose given 1 hour after the last MPTP dose on day 1.

[0202] Mice were subjected to kinematic gait analyses on day 7, using a Motorater (TSE Systems GmbH, Bad Homburg, Germany) test system. Animals were tested during their light cycle between 7 am and 8 pm. Before the movement and gait analysis sessions, mice were marked on 31 points of the body to facilitate data analysis of the captured videos. Movement was captured using a high-speed camera (300 fps) from three different directions, from below, and both sides.

[0203] The captured videos of each mouse were first converted to the software-readable format. To obtain raw data, the marked points of the body were tracked and each of the three directions were correlated. Thereafter, different gait patterns and movements were extracted using custom-made software developed by Charles River Discovery Research Service Finland. Gait pattern and movement analysis assessed 100 different parameters, including but not limited to stride time, swing time during a stride, speed, step width, stance and interlimb coordination. The data was analysed by using principal component analysis (PCA). The overall gait analysis was based on the PCA of all parameters for each mouse, with the obtained value showing the overall differences, measured as a “distance”, between control mice and MPTP, or MPTP plus ApoTf mice. Control mice (Controls) are set to a value of 0, with the “Distance from Control” shown for MPTP only mice (MPTP), or MPTP mice with subsequent ApoTf treatment (MPTP 4 ApoTf). Values are shown as mean+/−SEM (n=15).

TABLE-US-00002 Sequences The sequences referred to in the preceding text are outlined below in fasta format. Human Transferrin [UniProt Q06AH7] protein sequence SEQ ID NO: 1 VPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGP SVACVKKASYLDCIRAIAANEADAVTLDAGLVYDA YLAPNNLKPVVAEFYGSKEDPQTFYYAVAVVKKDS GFQMNQLRGKKSCHTGLGRSAGWNIPIGLLYCDLP EPRKPLEKAVANFFSGSCAPCADGTDFPQLCQLCP GCGCSTLNQYFGYSGAFKCLKDGAGDVAFVKHSTI FENLANKADRDQYELLCLDNTRKPVDEYKDCHLAQ VPSHTVVARSMGGKEDLIWELLNQAQEHFGKDKSK EFQLFSSPHGKDLLFKDSAHGFLKVPPRMDAKMYL GYEYVTAIRNLREGTCPEAPTDECKPVKWCALSHH ERLKCDEWSVNSVGKIECVSAETTEDCIAKIMNGE ADAMSLDGGFVYIAGKCGLVPVLAENYNKSDNCED TPEAGYFAVAVVKKSASDLTWDNLKGKKSCHTAVG RTAGWNIPMGLLYNKINHCRFDEFFSEGCAPGSKK DSSLCKLCMGSGLNLCEPNNKEGYYGYTGAFRCLV EKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYE LLCLDGTRKPVEEYANCHLARAPNHAVVTRKDKEA CVHKILRQQQHLFGSNVTDCSGNFCLFRSETKDLL FRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLRKC STSSLLEACTFRRP Human Lactoferrin [UniProt P02788] protein sequence SEQ ID NO: 2 GRRRRSVQWCTVSQPEATKCFQWQRNMRRVRGPPV SCIKRDSPIQCIQAIAENRADAVTLDGGFIYEAGL APYKLRPVAAEVYGTERQPRTHYYAVAVVKKGGSF QLNELQGLKSCHTGLRRTAGWNVPIGTLRPFLNWT GPPEPIEAAVARFFSASCVPGADKGQFPNLCRLCA GTGENKCAFSSQEPYFSYSGAFKCLRDGAGDVAFI RESTVFEDLSDEAERDEYELLCPDNTRKPVDKFKD CHLARVPSHAVVARSVNGKEDAIWNLLRQAQEKFG KDKSPKFQLFGSPSGQKDLLFKDSAIGFSRVPPRI DSGLYLGSGYFTAIQNLRKSEEEVAARRARVVWCA VGEQELRKCNQWSGLSEGSVTCSSASTTEDCIALV LKGEADAMSLDGGYVYTAGKCGLVPVLAENYKSQQ SSDPDPNCVDRPVEGYLAVAVVRRSDTSLTWNSVK GKKSCHTAVDRTAGWNIPMGLLFNQTGSCKFDEYF SQSCAPGSDPRSNLCALCIGDEQGENKCVPNSNER YYGYTGAFRCLAENAGDVAFVKDVTVLQNTDGNNN DAWAKDLKLADFALLCLDGKRKPVTEARSCHLAMA PNHAVVSRMDKVERLKQVLLHQQAKFGRNGSDCPD KFCLFQSETKNLLFNDNTECLARLHGKTTYEKYLG PQYVAGITNLKKCSTSPLLEACEFLRK Y188F Transferrin N-lobe mutant protein SEQ ID NO: 3 VPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGP SVACVKKASYLDCIRAIAANEADAVTLDAGLVYDA YLAPNNLKPVVAEFYGSKEDPQTFYYAVAVVKKDS GFQMNQLRGKKSCHTGLGRSAGWNIPIGLLYCDLP EPRKPLEKAVANFFSGSCAPCADGTDFPQLCQLCP GCGCSTLNQYFGFSGAFKCLKDGAGDVAFVKHSTI FENLANKADRDQYELLCLDNTRKPVDEYKDCHLAQ VPSHTVVARSMGGKEDLIWELLNQAQEHFGKDKSK EFQLFSSPHGKDLLFKDSAHGFLKVPPRMDAKMYL GYEYVTAIRNLREGTCPEAPTDECKPVKWCALSHH ERLKCDEWSVNSVGKIECVSAETTEDCIAKIMNGE ADAMSLDGGFVYIAGKCGLVPVLAENYNKSDNCED TPEAGYFAVAVVKKSASDLTWDNLKGKKSCHTAVG RTAGWNIPMGLLYNKINHCRFDEFFSEGCAPGSKK DSSLCKLCMGSGLNLCEPNNKEGYYGYTGAFRCLV EKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYE LLCLDGTRKPVEEYANCHLARAPNHAVVTRKDKEA CVHKILRQQQHLFGSNVTDCSGNFCLFRSETKDLL FRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLRKC STSSLLEACTFRRP Y95F/Y188F Transferrin N-lobe mutant protein SEQ ID 4 VPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGP SVACVKKASYLDCIRAIAANEADAVTLDAGLVYDA YLAPNNLKPVVAEFYGSKEDPQTFFYAVAVVKKDS GFQMNQLRGKKSCHTGLGRSAGWNIPIGLLYCDLP EPRKPLEKAVANFFSGSCAPCADGTDFPQLCQLCP GCGCSTLNQYFGFSGAFKCLKDGAGDVAFVKHSTI FENLANKADRDQYELLCLDNTRKPVDEYKDCHLAQ VPSHTVVARSMGGKEDLIWELLNQAQEHFGKDKSK EFQLFSSPHGKDLLFKDSAHGFLKVPPRMDAKMYL GYEYVTAIRNLREGTCPEAPTDECKPVKWCALSHH ERLKCDEWSVNSVGKIECVSAETTEDCIAKIMNGE ADAMSLDGGFVYIAGKCGLVPVLAENYNKSDNCED TPEAGYFAVAVVKKSASDLTWDNLKGKKSCHTAVG RTAGWNIPMGLLYNKINHCRFDEFFSEGCAPGSKK DSSLCKLCMGSGLNLCEPNNKEGYYGYTGAFRCLV EKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYE LLCLDGTRKPVEEYANCHLARAPNHAVVTRKDKEA CVHKILRQQQHLFGSNVTDCSGNFCLFRSETKDLL FRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLRKC STSSLLEACTFRRP Y426F/Y517F Transferrin C-lobe mutant protein SEQ ID NO: 5 VPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGP SVACVKKASYLDCIRAIAANEADAVTLDAGLVYDA YLAPNNLKPVVAEFYGSKEDPQTFYYAVAVVKKDS GFQMNQLRGKKSCHTGLGRSAGWNIPIGLLYCDLP EPRKPLEKAVANFFSGSCAPCADGTDFPQLCQLCP GCGCSTLNQYFGYSGAFKCLKDGAGDVAFVKHSTI FENLANKADRDQYELLCLDNTRKPVDEYKDCHLAQ VPSHTVVARSMGGKEDLIWELLNQAQEHFGKDKSK EFQLFSSPHGKDLLFKDSAHGFLKVPPRMDAKMYL GYEYVTAIRNLREGTCPEAPTDECKPVKWCALSHH ERLKCDEWSVNSVGKIECVSAETTEDCIAKIMNGE ADAMSLDGGFVYIAGKCGLVPVLAENYNKSDNCED TPEAGFFAVAVVKKSASDLTWDNLKGKKSCHTAVG RTAGWNIPMGLLYNKINHCRFDEFFSEGCAPGSKK DSSLCKLCMGSGLNLCEPNNKEGYYGFTGAFRCLV EKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYE LLCLDGTRKPVEEYANCHLARAPNHAVVTRKDKEA CVHKILRQQQHLFGSNVTDCSGNFCLFRSETKDLL FRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLRKC STSSLLEACTFRRP BDNF SEQ ID NO: 6 MFHQVRRVMTILFLTMVISYFGCMKAAPMKEANIR GQGGLAYPGVRTHGTLESVNGPKAGSRGLTSLADT FEHVIEELLDEDQKVRPNEENNKDADLYTSRVMLS SQVPLEPPLLFLLEEYKNYLDAANMSMRVRRHSDP ARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVL EKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKR HWNSQCRTTQSYVRALTMDS KKRIGWRFIRIDTS CVCTLT IKRGR GDNF SEQ ID NO: 7 MQSLPNSNGAAAGRDFKMKLWDVVAVCLVLLHTAS AFPLPAANMPEDYPDQFDDVMDFIQATIKRLKRSP DKQMAVLPRRERNRQAAAANPENSRGKGRRGQRGK NRGCVLTAIHLNVTDLGLGYETKEELIFRYCSGSC DAAETTYDKILKNLSRNRRLVSDKVGQACCRPIAF DDDLSFLDDNLVYHILRKHSAKRCGCI CNTF SEQ ID NO: 8 MAFTEHSPLTPHRRDLCSRSIWLARKIRSDLTALT ESYVKHQGLNKNINLDSADGMPVASTDQWSELTEA ERLQENLQAYRTFHVLLARLLEDQQVHFTPTEGDF HQAIHTLLLQVAAFAYQIEELMILLEYKIPRNEAD GMPINVGDGGLFEKKLWGLKVLQELSQWTVRSIHD LRFISSHQTGIPARGSHYIANNKKM PACAP SEQ ID NO: 9 HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK