COMPOSITIONS HAVING NEUROREGENERATIVE APPLICATIONS

Abstract

Pharmaceutical compositions containing transferrin and, or lactoferrin for use in promoting and or inducing the generation new neural cells in a patient that has suffered a neurodegenerative event arising from at least one of a traumatic brain injury, a non-traumatic brain injury, a spinal cord injury, a peripheral nerve injury, or peripheral neuropathy. 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 arising from at least one of a traumatic brain injury, a non-traumatic brain injury, a spinal cord injury, a peripheral nerve injury, or peripheral neuropathy, 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 traumatic brain injury or spinal cord injury is caused by at least one of road a traffic accident, an assault, a sporting collision, or an unprotected fall.

8. The method of claim 1, wherein the non-traumatic brain injury is caused by at least one of an ischaemic stroke, a haemorrhagic stroke, cerebral hypoxia, cerebral anoxia, consumption of chemical toxins, hydrocephalus, meningitis, or encephalitis.

9. The method of claim 1, wherein the neurodegenerative event arises from a stroke selected from the group consisting of a ischemic stroke and a haemorrhagic stroke.

10. The method of claim 1, further comprising administering a serum or plasma protein selected from the group consisting of Albumin, Alpha-1 Antitrypsin/Alpha-1 Proteinase Inhibitor, Antithrombin, polyclonal immunoglobulins, polyspecific immunoglobulins, C1 esterase inhibitor, Transthyretin, and combinations thereof to the patient in addition to the protein selected from transferrin, lactoferrin, and combinations thereof.

11-21. (canceled)

22. A method of stimulating neural cell development in a patient that has suffered a neurodegenerative event arising from at least one of a traumatic brain injury, a non-traumatic brain injury, a spinal cord injury, a peripheral nerve injury, or peripheral neuropathy, 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 2Z 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: v) Y188F mutant comprising the amino acid sequence set forth in SEQ ID NO: 3; vi) Y95F/Y188F mutant comprising the amino acid sequence set forth in SEQ ID NO: 4; vii) Y426F/Y517F mutant comprising the amino acid sequence set forth in SEQ ID NO: 5; and viii) 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 traumatic brain injury or spinal cord injury is caused by at least one of road a traffic accident, an assault, a sporting collision, or an unprotected fall.

29. The method of claim 22, wherein the non-traumatic brain injury is caused by at least one of an ischaemic stroke, a haemorrhagic stroke, cerebral hypoxia, cerebral anoxia, consumption of chemical toxins, hydrocephalus, meningitis, or encephalitis.

30. The method of claim 22, wherein the neurodegenerative event arises from a stroke selected from the group consisting of a ischemic stroke and a haemorrhagic stroke.

31. The method of claim 22, further comprising administering a serum or plasma protein selected from the group consisting of Albumin, Alpha-1 Antitrypsin/Alpha-1 Proteinase Inhibitor, Antithrombin, polyclonal immunoglobulins, polyspecific immunoglobulins, C1 esterase inhibitor, Transthyretin, and combinations thereof to the patient in addition to the protein selected from transferrin, lactoferrin, and combinations thereof.

32-42. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Additional features and advantages of the present invention will be made clearer in the appended drawings, in which:

[0112] FIGS. 1A to 1D show the induction of neurite outgrowth, and proliferation in SH-SY5Y cells and increased β-III-tubulin protein concentrations in response to apo-transferrin;

[0113] 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;

[0114] FIG. 3A plots the effect of deferoxamine mesylate at various concentrations relative to apo-transferrin on neurite outgrowth in SH-SY5Y cells;

[0115] FIG. 3B illustrates the efficacy of a transferrin mutant having reduced iron binding capacity on promoting neurite outgrowth in SH-SY5Y cells;

[0116] FIG. 4 plots the effect of various different proteins on neurite outgrowth in SH-SY5Y cells;

[0117] FIG. 5 plots the effect of IOX2, a prolyl hydroxylase inhibitor, on neurite outgrowth in SH-SY5Y cells;

[0118] FIGS. 6A & 6B illustrate the role of iron saturation on the efficacy of transferrin in promoting neurite outgrowth in SH-SY5Y cells;

[0119] 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;

[0120] FIG. 8 plots the effect of apo-transferrin in combination with the small molecule Y-27632 on neurite outgrowth in SH-SY5Y cells;

[0121] FIGS. 9A and 9B illustrate that apo-transferrin increases the amount of neurogenesis as measured by new neuroblasts (as defined by BrdU+/DCX+ cells) and newly formed mature neurons (as defined by BrdU+/NeuN+ cells) in an animal test model; and

[0122] FIGS. 10A to 10C demonstrate that treatment of animals with apo-transferrin after transient MCAo results in quicker recovery, better motor skills, and higher cognition compared to saline-treated mice.

DETALED EXAMPLES OF THE INVENTION

[0123] 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

[0124] 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 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

SH-SY5Y Cells

[0133] 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.

[0134] 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 SH-SY5Y cell line as a predictive model for brain injuries and neuropathies.

Neurogenesis

[0135] 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.

[0136] 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.

Peripheral Nerve Injury

[0137] Han et al. Berberine. Promotes Axonal Regeneration in Injured Nerves of the Peripheral Nervous System. J Med Food 15 (4) 2012, 413-417.

[0138] Gold et al. Nonimmunosuppressant FKBP-12 Ligand Increases Nerve Regeneration. EXPERIMENTAL NEUROLOGY 147, 269-278 (1997).

[0139] Kim et al. Protective effect of GCSB-5, an herbal preparation, against peripheral nerve injury in rats. Journal of Ethnopharmacology 136 (2011) 297-304.

[0140] Lesma et al. Glycosaminoglycans in Nerve Injury: I. Low Doses of Glycosaminoglycans Promote Neurite Formation. Journal of Neuroscience Research. 1996 46(5):565-71.

Diabetic Neuropathy

[0141] Hattangady and Rajadhyaksha. A brief review of in vitro models of diabetic neuropathy. Int J Diabetes Dev Ctries. 2009 October-December; 29(4): 143-149.

[0142] Vincent et al. Oxidative Stress and Programmed Cell Death in Diabetic Neuropathy. Ann. N.Y. Acad. Sci. 959: 368-383 (2002).

[0143] Shindo. Modulation of Basal Nitric Oxide-dependent Cyclic-GMP Production by Ambient Glucose, Myo-Inositol, and Protein Kinase C in SH-SY5Y Human Neuroblastoma Cells. J. Clin. Invest. Volume 97, Number 3, February 1996, 736-745.

[0144] Li et al. C-peptide enhances insulin-mediated cell growth and protection against high glucose-induced apoptosis in SH-SY5Y cells. Diabetes Metab Res Rev 2003; 19: 375-385.

Cancer Drug Induced Neuropathy

[0145] Rigolio et al. Resveratrol interference with the cell cycle protects human neuroblastoma SH-SY5Y cell from paclitaxel-induced apoptosis. Neurochemistry International 46 (2005) 205-211.

[0146] Donzelli et al. Neurotoxicity of platinum compounds: comparison of the effects of cisplatin and oxaliplatin on the human neuroblastoma cell line SH-SY5Y. Journal of Neuro-Oncology 67: 65-73, 2004.

[0147] Mannelli et al. Oxaliplatin-induced oxidativestressinnervoussystem-derived cellular models:Could it correlate with in vivo neuropathy? Free Radical Biology and Medicine 61 (2013) 143-150.

Organophosphate Induced Neuropathy (Insecticides, Chemical Warfare Compounds)

[0148] Hong et al. Neurotoxicity induced in differentiated SK-N-SH-SY5Y human neuroblastoma cells by organophosphorus compounds. Toxicology and Applied Pharmacology 186 (2003) 110-118.

[0149] Ehrich et al. Interaction of organophosphorus compounds with muscarinic receptors in SH-SY5Y human neuroblastoma cells. Journal of Toxicology and Environmental Health 1994 43(1):51-63.

Traumatic Brain Injury

[0150] Triyoso and Good. Pulsatile shear stress leads to DNA fragmentation in human SH-SY5Y neuroblastoma cell line. Journal of Physiology (1999), 515.2, pp. 355-365.

[0151] Song et al. Arctigenin Confers Neuroprotection Against Mechanical Trauma Injury in Human Neuroblastoma SH-SY5Y Cells by Regulating miRNA-16 and miRNA-199a Expression to Alleviate Inflammation. J Mol Neurosci (2016) 60:115-129.

[0152] Skotak et al. An in vitro injury model for SH-SY5Y neuroblastoma cells: Effect of strain and strain rate. Journal of Neuroscience Methods 205 (2012) 159-168.

[0153] Arun et al. Studies on blast traumatic brain injury using in-vitro model with shock tube. NeuroReport (2011) 22:379-384.

Ischemia

[0154] Miglio et al. Cabergoline protects SH-SY5Y neuronal cells in an in vitro model of ischemia. European Journal of Pharmacology 489 (2004) 157-165.

[0155] Duong et al. Multiple protective activities of neuroglobin in cultured neuronal cells exposed to hypoxia re-oxygenation injury. J. Neurochem. (2009) 108, 1143-1154.

[0156] Qiu et al. Enhancement of ischemia-induced tyrosine phosphorylation of Kv1.2 by vascular endothelial growth factor via activation of phosphatidylinositol 3-kinase. J. Neurochem. (2003) 10.104.

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

[0157] 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.

[0158] 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

[0159] 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.

[0160] 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. 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.

[0161] From FIG. 3 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.

[0162] The present inventors further sought to determine if a reduction of transferrin's iron-binding activity by mutation of the N-terminal iron-binding site was sufficient to mediate neurogenesis. Undifferentiated SH-SY5Y cells were treated as described in Example 1. Neurite 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 T racker 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.

[0163] 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.

[0164] 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 4) were obtained by cell culture expression from 293-6E cells.

[0165] 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 6xHIS 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 6xHIS tag and additional amino acids from the transferrin proteins. Following TEV cleavage, the transferrin proteins were separated from cleaved 6xHIS 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.

[0166] 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-SY5Y. 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-SY5Y 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

[0167] As the role of iron chelation in ApoTf's neurogenic ability was found to be unclear from Example 3 the present inventors determined whether other iron binding proteins can also mediate neurogenesis of SH-SY5Y cells.

[0168] 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. 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.

[0169] 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, p1449-1458. Holo-transferrin (HoloTf), the iron-saturated form of transferrin, was also unable to induce differentiation of the SH-SY5Y cells.

[0170] 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-SY5Y cells. This furthered the hypothesis that iron binding is not 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.

[0171] 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)

[0172] 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.

[0173] 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. 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 actions on PHD2. See Chowdhury et al., 2013. ACS Chem. Biol. Vol 8, p1488. IOX2 has an IC.sub.50 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).

[0174] 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.

[0175] 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

[0176] 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.

[0177] 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 Purity Saturation Sample Name (%) (%) 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

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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

[0182] 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), p351.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] Reviewing each of FIGS. 7A-7D it is apparent that each of the neurotrophic factors, and the peptide fragment induced differentiation of SH-SY5Y 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-SY5Y cells.

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

[0187] 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.

[0188] 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.

[0189] 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 intriguing finding.

Example 9: Apo-Transferrin Promotes New Neuroblast and Mature Neuron Formation in Brains of Animals with Transient MCAo

[0190] C57BL/6J mice (ca. 20 g) were anaesthetized under isoflurane and, after dissection, a 6.0 silicon-coated monofilament suture was inserted into the external carotid artery to occlude the middle cerebral artery (MCAo). The occlusion was carried out for 60 minutes under temperature control. Within two hours after removing the occlusion, animals were assessed on a 7-point ‘neuroscore’ scale to identify candidates with a visual demonstration of stroke on a scale from 0 (no observable deficit) to 6 (moribund)—the extension of the contralateral forepaw, the severity of circling, the loss of walking and consciousness were all taken into consideration. [0191] 0=no observable deficit [0192] 1=failure to extend the contralateral forepaw [0193] 2=mild circling behavior when picked up by the tail, <50% attempts to rotate to the contralateral side [0194] 3=mild consistent circling, >50% attempts to rotate to the contralateral side [0195] 4=consistent and strong circling, the mouse holds a rotation position for more than 1 to 2 seconds, with its nose almost reaching its tail [0196] 5=severe rotation with falling in a direction contralateral to the infarct, loss of walking or righting reflex, and [0197] 6=depressed level of consciousness, comatose, or moribund

[0198] Only animals with a ‘neuroscore’ of 4 or greater were considered for further testing (n=8-10 animals/group). At six hours post-occlusion, mice were injected daily with 350 mg/kg of Apo-transferrin (i.p.) or an equal volume of saline for a total of seven days, as well as with 50 mg/kg Bromodeoxyuridine (BrdU; Sigma-Aldrich Chemical, St. Louis, Mo.), both articles delivered by intraperitoneal administration. Weight changes were monitored daily.

[0199] At the times indicated in Example 9, brains were prepared for analysis by transcardial perfusion with ice cold heparinized saline (heparin at 2.5 IU/ml) in order to remove blood from the brains. Fresh brains were removed leaving the right cerebellum attached to whole left-brain hemisphere to help orientate the brain block in sectioning. The whole left hemisphere block was placed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 24 hours at +4° C., then cryoprotected in 30% sucrose in 0.1 M PB for 2-3 days on a shaker at +4° C. The brain was then blotted to remove excess liquid, placed on top of a vial cork and frozen on top of liquid nitrogen; the block was then stored at −80° C. until cryostat sections are obtained. Neurogenesis was evaluated by immunohistochemistry using antibodies against BrdU and Doublecortin (DCX) to quantify new neuroblasts or against BrdU and NeuN to quantify new neurons in the dentate gyrus of the brain.

[0200] FIG. 9A demonstrates that administration of ApoTf increases the number of neuroblasts over a two-week period, while FIG. 9B shows that after 4 weeks the number of newly formed mature neurons are higher in ApoTf-treated mice. These data suggest that the neuroblasts created early in neurogenesis may go on to differentiate further into new mature neurons. These results therefore suggest that apo-transferrin can promote aspects of neurogenesis above and beyond the neurogenesis that is normally induced in response to ischemic stroke.

Example 10: Apo-Transferrin Promotes Recovery, Motor Skills, and Cognition in a Mouse Model of Transient MCAo Stroke

[0201] Mice were prepared as indicated in Example 9 above and were subjected to assessments at 3 days and 1, 2, 3, 4 weeks post-MCAo, as indicated (n=8-10 animals/group). Motor coordination (i.e. equilibrium behavior and locomotor ability as a function of cortico-striatal function) was assessed using a rotarod test. Learning and memory capacity were measured using the NORT (novel object recognition test; for example, Anglada-Huguet et al., 2014, Molecular Neurobiology, vol. 49, pages 784-795; Denninger et al., 2018, J. Vis. Exp., vol. 141, e58593).

[0202] FIG. 10A shows that ApoTf significantly improves the rate at which the animals recover after MCA occlusion (MCAo) over the course of 3-14 days, and, possibly, longer. As described in Example 9, all animals in the study have an initial neuroscore of 4 or higher when assessed 2 hr after the MCAo. The percentage of animals that have no observable deficit (i.e. a neuroscore of ‘0’, as described in Example 9) are shown as a function of time after MCAo, as indicated on the x-axis.

[0203] FIG. 10B shows that animals treated with apo-transferrin have increased motor and balance skills after MCAo. The time that it takes an animal to fall (Latency to fall) from the rotarod is shown vs time after MCAo. The motor skills of the mice reflect those of the neuroscore in FIG. 10A, with an increased rate of improvement in motor/balance skills, as measured by the amount of time animals stayed on the rotarod apparatus. While the recovery as measured by neuroscore is the same in both groups by 4 weeks, the mice treated with apoTf have better overall abilities to remain on the rotarod. FIG. 10C provides evidence that administration of ApoTf increases learning recognition functions over at least a two-week period following administration of protein. The Discrimination (%) is a description of an animal's memory, as measured by the percentage of time in which an animal investigates newly presented objects compared to the time they investigate previously presented objects. Animals with better cognition and memory spend more time with the novel object due to memories of the object previously presented to the animal; this is shown as increased % Discrimination. The animals treated with apoTf have a higher ‘percent Discrimination’ as compared to saline-treated mice, suggesting that after MCAo, the apoTf-treated mice recover their cognitive abilities better.

[0204] Taken together, the data from FIGS. 10A to 10C suggest the neurogenesis promoted by apoTf translates to better motor ability and cognition within subjects.

Sequences

[0205] The sequences referred to in the preceding text are outlined below in fasta format.

TABLE-US-00002 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 ESYVKHQGLNKNINLDSADGMPVASTDOWSELTEA ERLQENLQAYRTFHVLLARLLEDQQVHFTPTEGDF HQAIHTLLLQVAAFAYQIEELMILLEYKIPRNEAD GMPINVGDGGLFEKKLWGLKVLQELSQWTVRSIHD LRFISSHQTGIPARGSHYIANNKKM PACAP SEQ ID NO: 9 HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRV KNK