Therapeutic methods using IGF-1 chimeric proteins

Abstract

Methods for treating a subject in need thereof using IGF-1 chimeric proteins while supporting normoglycemia are provided.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 482,904, filed February 2, 2023, and U.S. Application No. 63 / 588,481, filed October 6, 2023, each of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Reference This specification includes a Sequence Listing submitted herewith, which includes a file named 132463-011202.xml, created on February 1, 2024, and having the following size: 37,142 bytes, the contents of which are incorporated herein by reference.

[0003] FIELD OF THE DISCLOSURE Aspects of the present disclosure generally relate to pharmaceutical compositions comprising IGF-1 chimeric proteins and methods of using such IGF-1 chimeric proteins in subjects in need thereof. [Background technology]

[0004] Insulin-like growth factors (IGFs) constitute a family of proteins that have insulin-like and growth-stimulating properties. Summary of the Invention

[0005] An aspect of the present disclosure is a method of treating a subject in need thereof with an IGF-1 chimeric protein, the method comprising administering a solution comprising an effective amount of dextrose prior to administering the IGF-1 chimeric protein, the administration occurring over a period of about 48 hours, and administering a pharmaceutical composition comprising the effective amount of the IGF-1 chimeric protein, wherein the effective amount of dextrose supports normoglycemia. In some embodiments, the solution comprising the effective amount of dextrose is administered to a subject in need thereof by infusion. In some embodiments, the method comprises administering the solution comprising the effective amount of dextrose at a starting infusion rate of about 0.1 mL / kg / h. In some embodiments, the method further comprises adjusting the infusion rate in increments of +0.05 mL / kg / hr to +2 mL / kg / hr.

[0006] In some embodiments, the solution comprising an effective amount of dextrose comprises about 5% to about 10% dextrose (w / v) in water, hi some embodiments, the solution comprising an effective amount of dextrose comprises about 5% to about 10% dextrose (w / v) in a saline solution.

[0007] In some embodiments, the methods comprise administering a solution comprising an effective amount of dextrose about 1 minute to about 30 minutes prior to administration of the IGF-1 chimeric protein.

[0008] In some embodiments, the IGF-1 chimeric protein comprises a targeting domain comprising human annexin 5 (AnxV) or a variant thereof, and an activator domain comprising insulin-like growth factor (IGF-1) or a variant thereof. In some embodiments, the IGF-1 chimeric protein further comprises a peptide, wherein the peptide extends the half-life of the chimeric protein. In some embodiments, the targeting domain is a non-internalizing variant of annexin 5. In some embodiments, the IGF-1 chimeric protein is not substantially internalized by cells. In some embodiments, the IGF-1 chimeric protein comprises a non-internalizing variant of annexin 5, wherein the non-internalizing variant of annexin 5 comprises one or more mutations, wherein the one or more mutations comprise substitutions at a position corresponding to C316 and, optionally, one or more positions corresponding to R63, K70, K101, E138, D139, N160, and combinations thereof. In some embodiments, the activator domain of the chimeric protein is a variant of human insulin-like growth factor IGF-1 comprising one or more mutations, wherein the one or more mutations consist of substitutions at one or more positions corresponding to E3, Y24, Y31, Y60, and combinations thereof. In some embodiments, the IGF-1 chimeric protein further comprises a half-life modulator comprising a variant of human serum albumin (HSA) comprising one or more mutations, wherein the one or more mutations consist of substitutions at one or more positions corresponding to C58 and N527, and combinations thereof. In some embodiments, the IGF-1 chimeric protein comprises or consists of IGF1(E3R / Y31A)_lk7_HSA26-609(C58S / N527Q)_lk7_AnxV2-320(R63A / K70A / K101A / E138A / D139G / N160A / C316A). In some embodiments, the IGF-1 chimeric protein comprises or consists of the amino acid sequence set forth in SEQ ID NO:24.

[0009] In some embodiments, the methods involve administering an effective amount of an IGF-1 chimeric protein in a tapered manner over a period of 2 to 14 days or longer.

[0010] In some embodiments, administering an effective amount of an IGF-1 chimeric protein inhibits apoptosis.

[0011] In some embodiments, the pharmaceutical composition further comprises at least one physiologically acceptable carrier.

[0012] In some embodiments, the subject in need thereof is a human.

[0013] In some embodiments, the subject in need thereof has an acute CNS disorder.

[0014] In some embodiments, the subject in need thereof has an acute cardiovascular disorder.

[0015] In some embodiments, the subject in need thereof has acute radiation syndrome.

[0016] In some embodiments, the subject in need thereof has a chemical inhalation injury.

[0017] In some embodiments, the subject in need thereof has a dermatological disorder.

[0018] In some embodiments, the subject in need thereof has a traumatic injury.

[0019] In some embodiments, the subject in need thereof has a chronic neurodegenerative disease.

[0020] In some embodiments, the subject in need thereof is undergoing a procedure that carries a risk of iatrogenic injury.

[0021] In some embodiments, the subject in need thereof is undergoing organ transplantation treatment.

[0022] In some embodiments, the subject in need thereof is undergoing a cosmetic dermatology treatment.

[0023] Some embodiments of the present disclosure relate to an IGF-1 chimeric protein for use in a method of treating a subject in need thereof, the method comprising: administering a solution comprising an effective amount of dextrose prior to administering the IGF-1 chimeric protein, wherein the administration is for a period of about 48 hours; and administering a pharmaceutical composition comprising an effective amount of the IGF-1 chimeric protein, wherein the effective amount of dextrose supports normoglycemia.

[0024] Some embodiments of the present disclosure relate to dextrose for use in a method of treating a subject in need thereof using an IGF-1 chimeric protein, the method comprising: administering a solution comprising an effective amount of the dextrose prior to administering the IGF-1 chimeric protein, wherein the administration is over a period of about 48 hours; and administering a pharmaceutical composition comprising an effective amount of the IGF-1 chimeric protein, wherein the effective amount of dextrose supports normoglycemia.

[0025] Some embodiments of the present disclosure relate to a combination of an IGF-1 chimeric protein and dextrose for use in a method of treating a subject in need thereof, the method comprising: administering a solution comprising an effective amount of dextrose prior to administering the IGF-1 chimeric protein, wherein the administration is for a period of about 48 hours; and administering a pharmaceutical composition comprising an effective amount of the IGF-1 chimeric protein, wherein the effective amount of dextrose supports normoglycemia.

[0026] Some embodiments of the present disclosure relate to the use of an IGF-1 chimeric protein in the manufacture of a medicament for the treatment of a subject in need thereof, wherein a solution comprising an effective amount of dextrose is administered prior to administration of a pharmaceutical composition comprising an effective amount of an IGF-1 chimeric protein, and wherein administration of the solution is for a period of about 48 hours, and wherein the effective amount of dextrose supports normoglycemia.

[0027] Some embodiments of the present disclosure relate to the use of dextrose and an IGF-1 chimeric protein in the manufacture of a medicament for treating a subject in need thereof, wherein a solution comprising an effective amount of dextrose is administered prior to administering a pharmaceutical composition comprising an effective amount of an IGF-1 chimeric protein, and wherein the administration of the solution comprising the effective amount of dextrose is for a period of about 48 hours, and wherein the effective amount of dextrose supports normoglycemia.

[0028] Some embodiments of the present disclosure relate to the use of a combination of an IGF-1 chimeric protein and dextrose in the manufacture of a medicament for treating a subject in need thereof, wherein a solution comprising an effective amount of dextrose is administered prior to administering a pharmaceutical composition comprising an effective amount of an IGF-1 chimeric protein, and wherein the administration of the solution comprising the effective amount of dextrose is for a period of about 48 hours, and wherein the effective amount of dextrose supports normoglycemia.

[0029] Sequence Listing Description SEQ ID NO: 1 is the amino acid sequence of wild-type human IGF-1 (mature form).

[0030] SEQ ID NO: 2 is the amino acid sequence of a variant of the wild-type human IGF-1 variant containing the E3R and Y31A substitutions.

[0031] SEQ ID NO: 3 is the amino acid sequence of a variant of human IGF-1 (IGF-1 LONG).

[0032] SEQ ID NO: 4 is the amino acid sequence of a variant of human IGF-1 (IGF1 E3R).

[0033] SEQ ID NO: 5 is the amino acid sequence of a variant of human IGF-1 (IGF-1Des 1-3).

[0034] SEQ ID NO: 6 is the amino acid sequence of a variant of human IGF-1 (IGF-1 LR3).

[0035] SEQ ID NO: 7 is the amino acid sequence of a variant of human IGF-1 (IGF1 R37X).

[0036] SEQ ID NO: 8 is the amino acid sequence of a variant of human IGF-1 (IGF1 3X) having a deletion of residues 68-70.

[0037] SEQ ID NO: 9 is the amino acid sequence of wild-type human annexin A5 (AnxV).

[0038] SEQ ID NO: 10 is the amino acid sequence of amino acids 2-320 of wild-type annexin 5 and a variant of wild-type human annexin 5 containing the following substitutions: R63A, K70A, K101A, E138A, D139G, N160A, and C316A.

[0039] SEQ ID NO: 11 is the amino acid sequence of the non-internalizing variant of human annexin A5 (ni-AnxV).

[0040] SEQ ID NO: 12 is the amino acid sequence of wild-type human serum albumin (HSA).

[0041] SEQ ID NO: 13 is the amino acid sequence of human serum albumin variant mHSA (C34S, N503Q substitution).

[0042] SEQ ID NO: 14 is the amino acid sequence of human serum albumin variant mHSA7 (C34S, N503Q, E505G and V547A substitutions).

[0043] SEQ ID NO: 15 is the amino acid sequence of variant human serum albumin containing amino acids 26-609 of wild-type human serum albumin and the C58S and N527Q substitutions.

[0044] SEQ ID NO: 16 is the amino acid sequence of the peptide linker.

[0045] SEQ ID NO: 17 is the amino acid sequence of human transferrin (Tf).

[0046] SEQ ID NO: 18 is the amino acid sequence of human alphafetoprotein (AFP).

[0047] SEQ ID NO: 19 is the amino acid sequence of human vitamin D binding protein (VDBP).

[0048] SEQ ID NO: 20 is the amino acid sequence of human transthyretin (TTR).

[0100] SEQ ID NO: 21 is the amino acid sequence of the PAS motif.

[0101] SEQ ID NO: 22 is the amino acid sequence of an albumin binding domain human antibody (aldudAB).

[0049] SEQ ID NO: 23 is the amino acid sequence of the peptide linker lk7.

[0050] SEQ ID NO: 24 is the amino acid sequence of the chimeric protein scp776.

[0051] Sequence number 25 is the amino acid sequence of the chimeric protein IGF1(E3R / Y31A)_lk7_HSA26-609(C58S / N527Q)_lk7_AnxV2-320(R63A / K70A / K101A / E138A / D139G / N160A / C316S). [Brief explanation of the drawings]

[0052] [Figure 1] Figure 1 is a non-quantitative schematic showing the response of organ tissues to acute injury with or without treatment with scp776.

[0053] [Figure 2A] Figure 2A is a schematic diagram showing the interaction of scp776 with healthy or apoptotic cells.

[0054] [Figure 2B]FIG. 2B is a graph showing the pro-survival signal versus scp776 concentration in healthy and injured cells.

[0055] [Figure 3] Figure 3 is a graph showing plots of mean serum scp776 concentrations for three cohorts in the Phase 1A study SCP-CL-0001. The inset is a graph of the same data on a semi-log scale.

[0056] [Figure 4] FIG. 4 is a graph showing the area above 70 mg / dL of blood glucose (euglycemic area) calculated from the blood glucose versus time profiles of subjects in the Phase 1A study SCP-CL-0001 (-dextrose) and the Phase 1B study SCP-CL-0002 (+dextrose).

[0057] [Figure 5-1] Figures 5A and 5B are graphs showing the preclinical efficacy of scp776 in an NHP model of AIS. Figure 5A: Study PR0362-1 compared three groups: placebo (n = 12), low-dose (scp776 total 24 mg / kg; n = 10), and high-dose (scp776 total 32 mg / kg; n = 10). Dose-dependence was observed for all measured parameters. For simplicity, only the high-dose group is shown. Blood-brain barrier integrity (gadolinium imaging, p = 0.0325), lesion size (T2 MRI, p = 0.0164), and neuropathy (neuropathy score, p = 0.0323) are shown as group means ± SEM for the high-dose and placebo groups. Multiple comparisons of means (ANOVA with least significant difference tests) were performed across all three groups to determine significant effects. The value for monkeys that died before scheduled evaluation was considered the maximum observed value. Figure 5B: Survival curves for high-dose scp776 (n=10) and placebo (n=12) groups from study PR0362-1 are plotted. [Figure 5-2] Same as above.

[0058] [Figure 6]Figure 6 shows the patient flow.

[0059] [Figure 7] Figure 7 shows blood glucose traces (Example 1). Blood glucose traces are plotted as circles and colored according to individual mice (n=8). Averaged responses are plotted as blue diamonds and connected by a line.

[0060] [Figure 8] Figure 8 is a graph showing total dextrose infusion volume for healthy human subjects: Cohort 1: filled circles, Cohort 2: filled squares, Cohort 3: filled triangles, Cohort 4: open circles, Cohort 5: open squares, Cohort 6: open triangles.

[0061] [Figure 9] FIG. 9 shows that the pharmacological and systemic effects of the IGF-1 component of scp776 can be countered by dextrose supplementation.

[0062] [Figure 10] FIG. 10 is a table showing the Phase 1a SAD and Phase 1b MAD trials.

[0063] [Figure 11-1] Figures 11A and 11B are graphs and a table showing that supplemented dextrose reduces the frequency of hypoglycemic events. [Figure 11-2] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0064] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments of the disclosure only and is not intended to be limiting.

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0066] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0067] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0068] The terms "peptide," "polypeptide," and "protein" are used interchangeably to mean a polymeric sequence of at least two amino acids covalently joined by amide bonds (also referred to herein as peptide bonds).

[0069] As used herein, the term "target molecule" refers to any molecule associated with a tissue (e.g., an "at risk," diseased, or damaged tissue). "Target cell" means a cell to which a protein or a targeting domain thereof can specifically bind.

[0070] "Binding" or "specific binding" are used interchangeably herein to indicate that a protein (or its targeting polypeptide domain or its activator domain) exhibits a substantial affinity for a particular molecule or cell or tissue that possesses said molecule (e.g., a targeting domain exhibits substantial affinity for a target molecule, or an activator domain exhibits substantial affinity for a molecule associated with the surface of a cell, such as a growth factor receptor), and is said to occur when the protein (or its targeting polypeptide domain or its activator domain) is selective in that it has substantial affinity for a particular molecule and does not exhibit significant cross-reactivity to other molecules.

[0071] "Identity," as known in the art, is a relationship between two or more polypeptide or protein sequences and can be determined by comparing the sequences. In the art, "identity" also refers to the degree of sequence relatedness between polypeptides or proteins, and can be determined by the match between strings of such sequences. "Identity" can be readily calculated by any bioinformatics method known in the art.

[0072] The term "parent polypeptide" refers to a wild-type polypeptide whose amino acid sequence or nucleotide sequence is part of a publicly available protein database (e.g., EMBL Nucleotide Sequence Database, NCBI Entrez, ExPasy, Protein Data Bank, and the like).

[0073] The term "mutant polypeptide" or "polypeptide variant" refers to a form of a polypeptide whose amino acid sequence differs from that of its corresponding wild-type (parent), naturally occurring, or any other parent form. A mutant polypeptide may contain one or more mutations, e.g., substitutions, insertions, deletions, additions, etc., that result in the mutant polypeptide. Generally, variants are closely similar overall and, in many regions, identical to the reference polypeptide. As used herein, "variant" refers to a polypeptide that differs in sequence from the native protein but retains at least one function and / or therapeutic property described elsewhere herein or otherwise known in the art.

[0074] The term "corresponding to a parent peptide" is used to describe polypeptides of the present disclosure in which the amino acid sequence of the polypeptide differs from that of a corresponding parent polypeptide only by the presence of at least one amino acid variation. Typically, the amino acid sequences of the variant polypeptide and the parent polypeptide exhibit a high percentage of identity. In one example, "corresponding to a parent peptide" means that the amino acid sequence of the variant polypeptide has at least about 50% identity, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% identity, or at least about 99% identity with the amino acid sequence of the parent polypeptide. In another example, the nucleic acid sequence encoding the variant polypeptide has at least about 50% identity, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% identity, or at least about 99% identity to the nucleic acid sequence encoding the parent polypeptide.

[0075] The term "substantial identity" or "substantial similarity," as used herein, when referring to a nucleic acid or fragment thereof, indicates that the nucleotide sequence identity is at least about 95% to 99% of the sequence when optimally aligned with another nucleic acid (or its complementary strand), using appropriate nucleotide insertions or deletions. The term "substantial identity" or "substantial similarity," as used herein, when referring to a protein or fragment thereof, indicates that the amino acid sequence identity is at least about 95% to 99% of the sequence when optimally aligned.

[0076] The term "damaged cell" or "damaged tissue," as used herein, means and includes biological cells or tissues, such as, but not limited to, neurons, glia, or nervous tissue, that have been damaged or impaired by traumatic or chemical injury, ischemic tissue, cells or tissues damaged by any means that result in the interruption of normal blood flow to the tissue.

[0077] The term "therapeutically effective amount," as used herein, means an amount of a protein or substance that will elicit the biological or medical response of a tissue, system, animal, or human that is sought by a researcher, veterinarian, physician, or other clinician.

[0078] The terms "pharmaceutically acceptable" or "physiologically acceptable," as used herein, mean the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0079] The terms "targeting moiety," "targeting domain," "targeting polypeptide," or "targeting module" are used interchangeably herein and refer to a molecule that selectively localizes a chimeric protein to a particular tissue or region of the body. Localization may be mediated by specific recognition of molecular determinants, the molecular size of the targeting domain, ionic interactions, hydrophobic interactions, and the like. As used herein, the terms "therapeutic moiety," "activator domain," "activator polypeptide," "signaling arm," and "effector module" are used interchangeably herein and refer to any substance that is therapeutically effective, non-toxic, does not have cytotoxic effects, or is not detrimental to cells. Such substances may include, but are not limited to, growth factors.

[0080] A "patient" or "subject" is a mammal, preferably a human. The term "treating" (or "treat" or "treatment") means slowing, alleviating, or reversing the progression or severity of a symptom, disorder, condition, or disease.

[0081] The term "therapeutically effective amount" refers to an amount or dose of a chimeric protein described herein that, upon single or multiple administration to a patient, provides the desired treatment.

[0082] As used herein, the term "physiologically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, more specifically in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the bispecific fusion protein is administered. Physiologically acceptable carriers can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, or sesame oil). Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, and ethanol. The pharmaceutical composition, if desired, can also contain minor amounts of wetting or emulsifying substances, or pH buffering substances.

[0083] Like insulin, endogenous IGF-1 has a known blood glucose-lowering effect, which has been shown to be the dose-limiting toxicity of exogenously administered IGF-1. Endogenous IGF-1 shares approximately 50% of its amino acid sequence with insulin and has a wide range of systemic metabolic and homeostatic effects in the body. Its direct effect on glucose metabolism is mediated by increased pancreatic insulin secretion and increased insulin sensitivity, leading to increased glucose uptake by muscle tissue. When administered to patients with severe insulin resistance and diabetes, IGF-1 improves glucose levels, but the doses required for these therapeutic effects also result in significant systemic adverse events.

[0084] IGF-1 chimeric protein The chimeric proteins provided herein are capable of specifically binding to two or more different specific molecules. In some embodiments, the chimeric protein comprises a targeting domain with binding specificity for a first specific target molecule, an activator domain with binding specificity for a second target molecule, and a half-life modulator.

[0085] In some aspects, the activator domain has binding specificity for a cell surface tyrosine kinase receptor. In some embodiments, binding of the activator to the tyrosine kinase receptor activates an intracellular signaling pathway associated with cell survival. In some aspects, the activator domain has binding specificity for a receptor that regulates / promotes tissue regeneration.

[0086] In some embodiments, the targeting domain serves to target the chimeric protein to a target cell or tissue, while the activator domain serves to activate an intracellular signaling pathway associated with cell survival.

[0087] In some embodiments, the half-life modulator increases the half-life of the chimeric protein.

[0088] In some embodiments, the chimeric protein is a fusion protein having a targeting polypeptide bound or linked to a half-life modulator and an activator polypeptide, hi some embodiments, the engineered protein is a chimeric protein having a targeting polypeptide bound or linked to a half-life modulator and a growth factor or mutant growth factor.

[0089] In some embodiments, mutant growth factors (e.g., IGF-1 variants) are engineered to have reduced efficacy but retain the ability to activate the cognate growth factor receptor, hi some embodiments, wild-type growth factors can be used as activator domains.

[0090] The targeting domain is generally used to target the chimeric protein to a target cell. In some embodiments, the target cell is apoptotic. The binding of the targeting domain to its target molecule does not induce a significant biological effect in the target cell. The activator domain binds to a receptor on the cell surface. The binding of the activator domain to its receptor is intended to mediate a specific biological effect, such as activating an intracellular signaling pathway related to cell survival. In some embodiments, the binding of the activator domain to its receptor is intended to positively regulate the survival of the target cell or tissue. In particular, the activator domain of the chimeric protein can promote survival signals.

[0091] In some embodiments, the in vivo activity of a chimeric protein can be assessed by detecting a signaling change in a molecule regulated by the activator domain, including, but not limited to, the phosphorylation state of a cell surface receptor or a downstream mediator such as phosphorylated AKT or phosphorylated ERK (as detected by flow cytometry, immunofluorescence, ELISA, phospholabeling, Western analysis of treated tissues, or any other method known in the art). In some embodiments, a chimeric protein is functional in vivo if it induces a significant (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or more) change in the level, functional activity, or phosphorylation of the regulated molecule as detected by the assay.

[0092] Activator Domain An activator domain can be any polypeptide that detectably modulates the activity of a cellular network. In some embodiments, an activator domain can activate a signaling pathway by binding to a cell surface receptor. In some embodiments, a particular activator domain is a growth factor polypeptide or any agonist of the receptor. It will be apparent that such modulation may increase the activity of a cellular network, such as inducing cell proliferation, inducing cell growth, promoting cell survival, and / or inhibiting apoptosis.

[0093] During acute injury, cells have three possible fates: survival, apoptosis, or necrosis. Some injured cells can naturally avoid cell death through a mechanism called apoptotic escape. Promotion of apoptotic escape after acute injury results in improved cell survival and preservation of organ function. Growth factors have the ability to promote cell and tissue survival by promoting apoptotic escape.

[0094] The activator domain for a particular application may be selected based on the desired therapeutic effect, for example, an activator domain comprising IGF-1 (or a variant or fragment thereof) can be used to enhance survival and neuroprotection.

[0095] In some embodiments, the activator domain comprises a change in the amino acid sequence, the three-dimensional structure of the protein, and / or the activity of the protein relative to the wild-type form of the protein.

[0096] In some embodiments, the activator domain comprises or consists of a growth factor having an amino acid sequence modification relative to a wild-type growth factor (e.g., IGF-1) that reduces its binding to its native receptor (e.g., IGF-1 receptor), reduces its binding to a binding protein (e.g., an IGF binding protein), and / or reduces its activation of its native receptor (e.g., IGF-1 receptor). In some embodiments, the activator domain is a growth factor having an amino acid sequence modification that reduces binding to its native receptor (e.g., IGF-1 receptor) (e.g., by about 1-5%, 5-10%, 10%-20%, about 20%-40%, about 50%, about 40%-60%, about 60%-80%, about 80%-90%, 90-95%).

[0097] The growth factor polypeptide detectably modulates growth factor receptor activation. In some embodiments, the activator domain of the bispecific protein is a growth factor, variant, or fragment thereof that retains at least about 0.01% of wild-type biological activity. In some embodiments, the activator domain of the bispecific protein is a growth factor, variant, or fragment thereof that retains at least about 0.1%, at least about 1%, or at least about 10% of wild-type biological activity. In some embodiments, the activator domain of the bispecific protein is a growth factor, variant, or fragment thereof that retains between about 0.01% and about 0.1% of wild-type biological activity. In some embodiments, the activator domain of the bispecific protein is a growth factor, variant, or fragment thereof that retains between about 0.01% and about 1% of wild-type biological activity. In some embodiments, the activator domain of the bispecific protein is a growth factor, variant, or fragment thereof that retains between about 0.01% and about 1% of wild-type biological activity. In some embodiments, the activator domain of the bispecific protein is a growth factor, variant, or fragment thereof that retains between about 0.01% and about 10% of wild-type biological activity. In some embodiments, the activator domain of the bispecific protein is a growth factor, variant, or fragment thereof that retains between about 0.1% and about 1% of its wild-type biological activity. In some embodiments, the activator domain of the bispecific protein is a growth factor, variant, or fragment thereof that retains between about 0.1% and about 10% of its wild-type biological activity. In some embodiments, the activator domain of the bispecific protein is a growth factor, variant, or fragment thereof that retains between about 0.1% and about 10% of its wild-type biological activity. In some embodiments, biological activity can be determined by measuring activation of the corresponding growth factor receptor in appropriate cells. In some embodiments, activation may be assessed, for example, by measuring phosphorylation of the receptor kinase or downstream effector protein, such as, but not limited to, AKT, S6, ERK, JNK, or mTOR.

[0098] Insulin-like growth factors (IGFs) and their derivatives Insulin-like growth factors (IGFs) constitute a family of proteins with insulin-like and growth-stimulating properties. IGF human IGF-1 is a 70-amino acid basic peptide, each with the protein sequence set forth in SEQ ID NO: 1. IGF-1 and extracellular tyrosine kinase receptors (e.g., IGF-1 receptor) are important for cellular processes such as cell proliferation and survival. Binding of IGF-1 or its variants to the IGF-1 receptor stimulates kinase activity, phosphorylating multiple substrates, thereby initiating a signal cascade. The chimeric proteins disclosed herein may retain the ability to signal through extracellular receptors, such as the IGF-1 receptor. The activator domain of IGF-1 stimulates cell proliferation and survival through activation of the AKT pathway. Upon binding of IGF-1 to the IGF-1 receptor, the tyrosine kinase phosphorylates tyrosine residues on two major substrates, IRS-1 and Shc, which subsequently signal through the Ras / Raf and PI 3-kinase / AKT pathways.

[0099] The interaction of IGF-1 (and IGF-2) with the IGF-1 receptor is regulated by IGF-binding proteins (IGFBPs). All six IGFBPs (especially IGFBP5) have shown inhibition of IGF action, although in some cases stimulatory effects have also been observed. At least 99% of IGFs in the circulation are normally bound to IGFBPs.

[0100] In some embodiments, the activator domain is a variant of human IGF-1 or a fragment thereof. In some embodiments, the variant of IGF-1 or a fragment thereof can maintain selectivity for the IGF-1 receptor.

[0101] In some embodiments, the IGF-1 variant is modified to have reduced binding to IGF-1 binding proteins (IGFBPs) compared to wild-type IGF-1, but maintains its ability to activate the AKT pathway. In some embodiments, the IGF-1 variant can activate the IGF-1 receptor with reduced effects on non-target cells, as assessed by pAKT EC50. EC50 is defined as the concentration required to reach half of the maximum level of pAKT signaling.

[0102] In some embodiments, the IGF-1 variant comprises a substitution at one or more tyrosine residues. In some embodiments, the IGF-1 variant comprises one or more substitutions at positions Y24, Y31, and Y60. In an exemplary embodiment, the IGF-1 variant may comprise a single tyrosine substitution at positions Y31, or Y24, or Y60. In an exemplary embodiment, the IGF-1 variant may comprise a single tyrosine substitution at positions Y24 and Y31, Y24 and Y60, Y31 and Y60, or Y24 and Y60. In another exemplary embodiment, the IGF-1 variant may comprise one or more of the following substitutions relative to wild-type IGF-1: Y24L, Y31A, and Y60L. For example, the IGF-1 variant may comprise a Y24L substitution and a Y31A substitution, or the IGF-1 variant may comprise a Y24L substitution, a Y31A substitution, and a Y60L substitution. In some embodiments, one or more tyrosine residues (Y24, Y31, Y60, or a combination thereof) may be substituted with a short-chain aliphatic amino acid. In some embodiments, one or more tyrosine residues (Y24, Y31, Y60, or a combination thereof) may be substituted with a polar amino acid. In some embodiments, one or more tyrosine residues (Y24, Y31, Y60, or a combination thereof) may be substituted with leucine, alanine, isoleucine, serine, threonine, or any other amino acid.

[0103] In some embodiments, the IGF-1 variant comprises a substitution of Arg for Glu, Lys, Met, Val, Ala, Leu, Ile, Gly, Ser, or Thr at position 3 of the polypeptide. In some embodiments, the IGF-1 variant comprises a substitution of Arg for Glu at position 3 of the polypeptide (E3R).

[0104] In some embodiments, the IGF-1 variant comprises substitutions at positions 3 and 31. For example, the IGF-1 variant comprises an E3R and a Y31A substitution. In some embodiments, the activator domain has an amino acid sequence having SEQ ID NO:2.

[0105] In some embodiments, the activator domain is a derivative of human IGF-1 comprising one or more of the following modifications: an N-terminal 13-residue extension (IGF-1 LONG), a deletion of amino acids 1-3 (Des-1-3), a substitution of Arg for Glu at position 3 of the polypeptide (E3R), the absence of arginine at position 37 (R37X), a deletion of amino acids 68-70 (3X), an N-terminal 13-residue extension and a substitution of Arg for Glu at position 3 of the wild-type polypeptide (LR3), a substitution of one or more tyrosine residues (Y24, Y31, Y60 or combinations thereof) (e.g., a Y24L, Y31A, Y60L substitution or combinations thereof).

[0106] In some embodiments, the activator domain is a variant of human IGF-1 that includes a mutation (eg, substitution, deletion) at one or more residues 24-37.

[0107] In some embodiments, the activator domain is a derivative of human IGF-1 and comprises an N-terminal 13-residue extension (also called IGF-1 LONG, SEQ ID NO:3), an E3R mutation (SEQ ID NO:4), or a combination thereof (LONG E3R, also called LR3, SEQ ID NO:6). In some embodiments, the IGF-1 variant comprises an E3R substitution, an N-terminal 13-residue extension, a deletion of amino acids 1-3 ((Des1-3), SEQ ID NO:5), or a combination thereof, which reduces binding of the activator domain to IGF-binding proteins present in serum and other body fluids.

[0108] In some embodiments, the activator domain is a derivative of human IGF-1 and includes one or more of the following modifications: an N-terminal 13-residue extension (SEQ ID NO: 3), a deletion of amino acids 1-3 (SEQ ID NO: 5), a substitution of Arg for Glu at position 3 of the polypeptide (SEQ ID NO: 4), the absence of arginine at position 37 (R37X, SEQ ID NO: 7), a deletion of amino acids 68-70 (3X, SEQ ID NO: 8), or an N-terminal 13-residue extension and a substitution of Arg for Glu at position 3 of the wild-type polypeptide (SEQ ID NO: 6).

[0109] Bispecific proteins containing variants of IGF-1 described herein (e.g., E3R, IGF-1 LONG, IGF-1 LONG E3R (referred to as IGF-1(LR3) or IGF1 Des1-3) are believed to have reduced affinity of IGF binding proteins relative to wild-type IGF-1. In some embodiments, the IGF-1 variants of the bispecific proteins described herein can activate signal activation pathways but have a substantially reduced interaction with IGF-1 binding proteins relative to wild-type IGF-1.

[0110] In some embodiments, the IGF-1 variant can be modified by glycosylation of one or more glycosylation sites present in the IGF-1 variant.

[0111] In some embodiments, chimeric proteins containing an IGF-1 variant described herein have an effect on non-target cells that is less than the effect of wild-type IGF-1 on non-target cells.

[0112] Certain activator domains that bind to growth factor receptors are provided herein in SEQ ID NOs: 1-8.

[0113] Additional peptide sequence modifications can include mutations, deletions, substitutions, or derivatizations of the amino acid sequence of the sequences disclosed herein, so long as the peptide has substantially the same activity or function as the unmodified peptide. In particular, the modified peptide will retain an activity or function associated with the unmodified peptide, and the modified peptide will generally have an amino acid sequence that is "substantially homologous" to the amino acid sequence of the unmodified sequence.

[0114] In some embodiments, an IGF-1 variant may have an amino acid sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identity to an amino acid sequence provided in SEQ ID NO: 1-8. In some embodiments, an IGF-1 variant may have an amino acid sequence having about 85% to about 90%, about 90% to about 95%, about 95% to about 98%, about 98% identity to about 99% identity to an amino acid sequence provided in SEQ ID NO: 1-8. In some embodiments, an IGF-1 variant may comprise 10, 20, 30, 40, 50, 60, or more contiguous amino acids of any one of the amino acids of SEQ ID NO: 1-8. In some embodiments, an IGF-1 variant may have the amino acid sequence set forth in any one of SEQ ID NO: 1-8. In some embodiments, the IGF-1 variant may have the amino acid sequence set forth in any one of SEQ ID NOs: 2-8. In some embodiments, the IGF-1 variant may have the amino acid sequence set forth in SEQ ID NO: 2.

[0115] In some embodiments, the bispecific protein comprises an activator domain having a growth factor variant, such as an IGF-1 variant, selected to confer an EC50 that is at least one order of magnitude lower in damaged tissue than in healthy tissue. For example, the bispecific protein domain comprises a growth factor variant, such as an IGF-1 variant, that has an EC50 in damaged tissue that is at least 10-fold lower, at least 15-fold lower, at least 20-fold lower, at least 25-fold lower, at least 30-fold lower, at least 35-fold lower, at least 40-fold lower, at least 45-fold lower, at least 50-fold lower, at least 55-fold lower, at least 60-fold lower, at least 65-fold lower, at least 70-fold lower, at least 75-fold lower, at least 80-fold lower, at least 85-fold lower, at least 90-fold lower, at least 95-fold lower, at least 100-fold lower, or at least 110-fold lower.

[0116] In some embodiments, the bispecific protein containing an IGF-1 variant has a lower half maximal effective concentration (EC50) in injured tissue than in healthy tissue. In some embodiments, the bispecific protein containing an IGF-1 variant has a half maximal effective concentration (EC50) in injured tissue than in healthy tissue that is at least 10-fold lower, at least 15-fold lower, at least 20-fold lower, at least 25-fold lower, at least 30-fold lower, at least 35-fold lower, at least 40-fold lower, at least 45-fold lower, at least 50-fold lower, at least 55-fold lower, at least 60-fold lower, at least 65-fold lower, at least 70-fold lower, at least 75-fold lower, at least 80-fold lower, at least 85-fold lower, at least 90-fold lower, at least 95-fold lower, at least 100-fold lower, or at least 110-fold lower.

[0117] In some embodiments, chimeric proteins comprising such variant growth factors provided herein have greater specificity for targeting damaged tissue.

[0102] target molecule In some aspects, the target molecule is exposed or concentrated on the outside of the target cell. In some embodiments, the target molecule is associated with damaged cells, early apoptotic cells, or apoptotic cells, and the target molecule is present intracellularly in viable or undamaged cells and exposed to the extracellular space in damaged cells. Such molecules include, for example, molecules exposed in cells undergoing necrosis (such as DNA) or apoptosis (such as phosphatidylserine), myosin (including its tissue-specific subtypes), ICAM-1, or P-selectin. However, in other embodiments, the target molecule is a molecule present or concentrated on the surface of diseased or dysfunctional cells or tissues compared to levels detected in healthy or functional cells or tissues. In some embodiments, the target cell is not a tumor or cancer cell.

[0103] Cells are enclosed by a plasma membrane (or cell membrane) that contains a lipid bilayer. The cell membrane may be considered to have a surface facing the cytosol (the cytosolic side or interior of the cell) and a surface facing the outside of the cell, or the extracellular space. The movement of anionic phospholipids across the bilayer from the inner to the outer leaflet of the plasma membrane occurs during apoptosis. Anionic phospholipid-binding proteins, such as annexin A5, synaptotagmin I, or lactadherin, can be used to detect the presence of phosphatidylserine on the outer leaflet of the plasma membrane. Phosphatidylserine is a phospholipid that is normally restricted to the cytosolic side of the membrane in viable or undamaged cells, but becomes exposed to the outer cell surface or extracellular space in damaged or apoptotic cells.

[0104] In some embodiments, the target molecule is an "ischemia-associated molecule." An "ischemia-associated molecule" is any molecule that is detected at levels significantly higher (e.g., at least 1.5-fold higher, at least 2-fold higher, at least 3-fold higher, at least 4-fold higher, at least 5-fold higher) following ischemia or hypoxia (resulting in hypoxia) than in cells of the same tissue that has not undergone the ischemic event (i.e., the molecule is specific or enriched in post-ischemic tissue). Ischemia occurs when blood flow is insufficient to supply sufficient oxygen, resulting in tissue hypoxia (reduced oxygen) or, the most severe form of hypoxia, anoxia (lack of oxygen), ultimately leading to tissue necrosis and apoptosis.

[0105] Targeted Domains In some embodiments, the targeting domain has specific binding affinity to a target molecule associated with a tissue (e.g., an ischemia-associated molecule). In some embodiments, the targeting domain has specific binding affinity to a target molecule present on the surface of early apoptotic cells. The targeting domain may be any polypeptide sequence that performs this function. In some embodiments, binding of the targeting domain to a target molecule does not have or modulate biological activity. As used herein, "biological activity" refers to a defined, known activity that results from exposing a molecule to a domain of a protein.

[0106] In some embodiments, the targeting domain may be a non-antibody peptide, a fragment thereof, or a variant thereof that has binding affinity to the target molecule, although in other embodiments, the targeting polypeptide domain comprises one or more antibody variable regions (e.g., scFv).

[0118] Annexin A5 and its variants In some embodiments, the targeting domain comprises an annexin, a variant thereof, or a fragment thereof. The term "annexin" refers to any protein capable of binding to phospholipids, particularly phosphatidylserine (PS), and to members of the annexin family. In some embodiments, the annexin is annexin A5, although other annexins can be used as well. In some embodiments, the targeting domain is human annexin A5, a functional fragment thereof, or a variant thereof. A variant of annexin A5 contains at least one amino acid, at at least one position, that is not found in the parent wild-type annexin A5 polypeptide (SEQ ID NO: 9). The annexin variant may contain one or more amino acid substitutions, deletions, additions, or a combination thereof, where the amino acid substitutions, deletions, or additions do not substantially affect the ability of the annexin A5 variant of the chimeric protein to bind to at least one phospholipid, such as PS. In some embodiments, an annexin A5 variant may have an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 98% identical, or at least about 99% identical to the amino acid sequence provided in SEQ ID NO: 9. In some embodiments, an annexin A5 variant may comprise 50, 80, 100, 110, 200, 300, or more contiguous amino acids that are at least about 85%, at least about 90%, at least about 95%, at least about 98% identical, or at least about 99% identical to the amino acids of SEQ ID NO: 9.

[0119] In some embodiments, annexin A5 variants are modified to substitute serine or alanine for the cysteine ​​at position 315 (corresponding to C316) to reduce dimer formation. For example, cysteine ​​can be substituted with alanine or serine. As used herein, the term "corresponding" is used to indicate the position / identity of an amino acid residue in a polypeptide (e.g., annexin A5). Those skilled in the art will recognize that for simplicity, a standard numbering system (based on wild-type annexin A5) is utilized herein; therefore, for example, an amino acid "corresponding" to a residue at position 316 need not be the actual 316th amino acid in a particular amino acid chain, but rather corresponds to the residue found at position 316 in annexin A5 prior to post-translational removal of the N-terminal methionine; those skilled in the art will readily understand how to identify the corresponding amino acid. In particular, note that the amino acid sequence of wild-type annexin A5 (SEQ ID NO: 9) does not begin with a methionine because the methionine residue is cleaved during processing.

[0120] In some embodiments, the annexin A5 variant has a mutated amino acid sequence that reduces cellular internalization of annexin A5 or a chimeric protein comprising the annexin A5 variant but maintains its binding affinity to phosphatidylserine (PS). In some embodiments, the annexin A5 variant or a chimeric protein comprising the annexin A5 variant has binding affinity to phosphatidylserine and is not internalized into cells or is internalized at a slower rate than wild-type annexin A5. In some embodiments, the targeting domain is a non-internalizing variant of annexin A5 (also referred to as ni-annexin A5 or ni-AnxV, SEQ ID NO: 11). In some embodiments, the annexin A5 variant has the amino acids set forth in SEQ ID NO: 10. In some embodiments, the non-internalizing mutant of annexin A5 has an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% identical to the amino acid sequence provided in SEQ ID NO: 11. In some embodiments, a non-internalizing mutant of annexin A5 can have an amino acid sequence having about 85% to about 90%, about 90% to about 95%, about 95% to about 98%, or about 98% to about 99% identity to the amino acid sequence provided in SEQ ID NO: 11. In some embodiments, an annexin A5 variant can comprise 50, 80, 100, 110, 200, 300, or more contiguous amino acids of any one of the amino acids of SEQ ID NO: 11. Any variation of annexin A5 that is substantially non-internalizable is also contemplated.

[0121] It should be understood that a non-internalizing variant of annexin A5 confers an extended half-life to the chimeric protein compared to a chimeric protein containing wild-type A5. In some embodiments, a variant of annexin A5 that does not undergo substantial internalization, or a chimeric protein containing a variant of annexin A5 that does not undergo substantial internalization, may have an extended half-life of 1.1 to 1.2, 1.1 to 1.3, 1.1 to 1.4, 1.1 to 1.5, 1.1 to 1.6, 1.1 to 1.7, 1.1 to 1.8, 1.1 to 1.9, 1.1 to 2, or more, compared to wild-type annexin A5 or a chimeric protein containing wild-type annexin A5.

[0122] The terms "non-internalized" and "not substantially internalized," as used herein, refer to the lack of a substantial amount of internalization of the chimeric proteins disclosed herein. For example, the phrase "not substantially internalized" may be understood to mean that less than 50% of the chimeric protein is internalized by cells to which the chimeric protein binds, or less than 25% of the chimeric protein is internalized by cells to which the chimeric protein binds, or less than 10% of the chimeric protein is internalized by cells to which the chimeric protein binds, or less than 5% of the chimeric protein is internalized by cells to which the chimeric protein binds, or less than 3% of the chimeric protein is internalized by cells to which the chimeric protein binds, or less than 1% of the chimeric protein is internalized by cells to which the bispecific protein binds.

[0123] In some embodiments, a non-internalizing mutant of annexin A5 can have an amino acid sequence that is at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to human annexin A5. In some embodiments, a non-internalizing variant of annexin A5 comprises a substitution at position 315 (corresponding to C316) in which the cysteine ​​residue is replaced with serine (Ser), alanine (Ala), leucine (Leu), phenylalanine (Phe), methionine (Met), or tryptophan (Trp). In some embodiments, a non-internalizing mutant of annexin A5 can have an amino acid sequence that is at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to human annexin A5. In some embodiments, a non-internalizing variant of annexin A5 comprises a substitution at position 315 (corresponding to C316), where the cysteine ​​residue is replaced with serine (Ser) or alanine (Ala). In some embodiments, a non-internalizing mutant of annexin A5 may have an amino acid sequence that is at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to human annexin A5 modified by substituting serine or alanine for the cysteine ​​at position 315 (corresponding to C316).

[0124] In some embodiments, annexin A5 or annexin A5 variants (e.g., variants with a substitution at position C316) are modified to include one or more substitutions at the following positions: R62, K69, K100, E137, D138, N159, L313 (corresponding to R63, K70, K101, E138, D139, N160, L314 in wild-type human annexin A5).

[0125] In some embodiments, the annexin A5 variant is R62A, R62E, R62D, R62M, R62L, R62I, R62Y (corresponding to R63A, R63E, R63D, R63M, R63L, R63I, R63Y in wild-type human annexin A5); K69A, K69E, K69D, K69M, K69L, K69I, K69Y (corresponding to K70A, K70E, K70D, K70M, K70L, K70I, K70Y of wild-type human annexin A5); E137A, E137K, E137R, E137M, E137L, E137I, E137Y (corresponding to E138A, E138K, E138R, E138M, E138L, E138I, E138Y in wild-type human annexin A5); D138G, D138K, D138R, D138M, D138L, D138I, D138Y (corresponding to D139G, D139K, D139R, D139M, D139L, D139I, D139Y in wild-type human annexin A5); N159A, N160M, N160L, N160I, N160V, and N160Y (corresponding to wild-type human annexin A5 N160A, N160M, N160L, N160I, N160V, and N160Y) L313E, L313D, L313K, L313R, L313H, L313Q, L313N, L313Y (corresponding to L314E, L314D, L314K, L314R, L314H, L314Q, L314N, L314Y in wild-type human annexin A5); or any combination of the above Includes.

[0126] In some embodiments, the annexin A5 or annexin A5 variant comprises one or more substitutions at positions D143 and / or E227. D142G, D142A, D142K, or D142R (corresponding to D143G, D143A, D143K, or D143R) substitution and / or Replaces E226G, E226A, E226K, or E226R (compatible with E227G, E227A, E227K, and E227R) Includes.

[0127] In some embodiments, annexin A5 or annexin A5 variants (e.g., having substitutions at C316, D143 and / or E227) are modified to include one or more of the following substitutions: R62A, K69A, K100A, E137A, D138G, N159A, L313E (corresponding to R63A, K70A, K101A, E138A, D139G, N160A, L314E). For example, annexin A5 having sequence number 9 can be modified to have a C315A or C315S substitution (corresponding to C316A or C316S in wild-type annexin A5) and one or more of the following substitutions: R62A, K69A, K100A, E137A, D138G, N159A, L313E (corresponding to R63A, K70A, K101A, E138A, D139G, N160A, L314E in wild-type annexin A5).

[0128] In some embodiments, human annexin A5 (sequence number 9) is modified to include one or more of the following substitutions: R62A, K69A, K100A, E137A, D138G, N159A, D143N, E227A, C315S, or C315A (corresponding to R63A, K70A, K101A, E138A, D139G, D144N, N160A, E228A, C316S, or C316A in wild-type annexin A5).

[0129] In some embodiments, the targeting domain is engineered annexin A5 to have R63A, K70A, K101A, E138A, D139G, N160A, and C316A or C316S substitutions relative to wild-type annexin A5. For example, the targeting domain may have the amino acid sequence of SEQ ID NO: 10.

[0130] In some embodiments, annexin A5 variants contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more substitutions in different regions to further reduce internalization of annexin within cells. For example, annexin A5 variants may include R62A and K69A, R62A and K100A, R62A and E137A, R62A and D138G, R62A and N159A, R62A and K69A and K100A, R62A and K69A and E137A, R62A, K69A and K100A, R62A, K69A, K100A, and E137A, etc.

[0131] The annexin variant may further comprise one or more amino acid substitutions, deletions, or additions, which do not substantially affect the ability of the annexin A5 variant of the chimeric protein to bind to at least one phospholipid, such as PS.

[0132] Native polypeptides can be used as targeting domains, however, it will be apparent that polypeptides having portions of such native sequences as well as modified sequences may also be used, provided that such polypeptides retain the ability to bind to the target molecule with an appropriate binding affinity (Kd), as described in detail below.

[0133] Antibody targeting domain: In some embodiments, anti-phosphatidylserine antibodies can be used as targeting domains. As used herein, the term "antibody" includes, but is not limited to: (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab)2 and F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) scFv fragments consisting of the VL and VH domains of a single arm of an antibody; (v) dAb fragments consisting of the VH domain; and (vi) isolated complementarity-determining regions (CDRs). Such antibodies can be produced from intact antibodies using methods known in the art, or can be produced recombinantly using standard recombinant DNA and protein expression techniques.

[0134] Targeting domain binding In some embodiments, the chimeric protein binds to the target molecule. -6 Less than M, preferably 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 K less than M d Combine with.

[0135] Half-Life Modulators Those skilled in the art will understand that proteins used in therapeutic applications may not exhibit optimal serum half-lives due to their relatively low molecular weight. In some therapeutic applications, it may therefore be desirable to extend the half-life of the protein. In some embodiments, the chimeric protein is conjugated, functionally associated with, or fused to a half-life modulator to accumulate the chimeric protein at diseased, damaged, or injured sites in an organ. Preferably, the half-life modulator is a non-immunogenic polypeptide.

[0136] For example, short half-life is the most limiting feature of wild-type growth factors as therapeutic agents.Intravenously administered IGF-1 has a serum half-life of less than 1 hour in humans.The extended half-life of the chimeric protein disclosed herein compared to IGF-1 allows, for example, 1) equivalent effect with less frequent administration; 2) equivalent exposure at a lower dose; 3) lower Cmax at equivalent exposure level, thereby reducing the risk of Cmax-related toxicity.

[0137] In some embodiments, the half-life modulator may extend the half-life of the chimeric protein in vivo. For example, the half-life of the chimeric protein comprising the half-life modulator is about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, or more. For example, the half-life of the chimeric protein may be about 8 hours or more when tested in cynomolgus monkeys. In some embodiments, the half-life of the chimeric protein comprising the half-life modulator is about 24 hours or more. In some embodiments, the half-life of the chimeric protein comprising the half-life modulator is about 1 week or more.

[0138] In some embodiments, the half-life modulator is non-immunogenic in humans.

[0139] In some embodiments, the half-life modulator is a polypeptide that interacts with cellular machinery that promotes escape from the lysosomal degradation pathway (eg, FcRn receptor-mediated recycling).

[0140] In some embodiments, the half-life modulator is designed to extend the half-life of the chimeric protein through binding to a serum component, such as human serum albumin (HSA), which is the most abundant protein in blood and has been shown to be safe in humans.

[0141] In some embodiments, the half-life modulator is an HSA variant. In some embodiments, the half-life modulator comprises at least 100 contiguous amino acids that are at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the wild-type human serum albumin amino acid sequence (wtHSA, SEQ ID NO: 12). In some embodiments, the half-life modulator comprises at least 200 contiguous amino acids that are at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the wild-type human serum albumin amino acid sequence. In some embodiments, the half-life modulator comprises at least 300 contiguous amino acids that are at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a wild-type human serum albumin amino acid sequence, hi some embodiments, the half-life modulator comprises at least 400 contiguous amino acids that are at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a wild-type human serum albumin amino acid sequence. In some embodiments, the half-life modulator comprises at least 500 contiguous amino acids that are at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a wild-type human serum albumin amino acid sequence.

[0142] In some embodiments, the HSA variant may have one or more of the following substitutions: Cysteine ​​C58 may be substituted, for example, with serine (C58S), alanine (C58A), asparagine (C58N), leucine (C58L), or glutamine (C58Q); Lysine K420 may be substituted, for example, with glutamic acid (K420E), aspartic acid (K420D), leucine (K420L) or methionine (K410M); Asparagine N527 may be substituted, for example, with glutamine (N527Q), aspartic acid (N527D), histidine (N527H), or tyrosine (N527Y); Glutamic acid E505 may be substituted with, for example, glycine (E505G), alanine (E505A), leucine (E505L), lysine (E505K), valine (E505V), isoleucine (E505I), methionine (E505M), or glutamine (E505Q); Valine V547 may be substituted with, for example, alanine (V547A), glycine (V547G), leucine (V547L), lysine (V547K), isoleucine (V547I), methionine (V547M); Asparagine N503 may be substituted with, for example, glutamine (N527Q), aspartic acid (N503D), histidine (N503H), or tyrosine (N503Y), or glutamine (V547Q).

[0143] In some embodiments, the HSA variant has amino acids 26-609 and may have one or more of the following substitutions: Cysteine ​​C58 may be substituted, for example, with serine (C58S), alanine (C58A), asparagine (C58N), leucine (C58L), or glutamine (C58Q); Lysine K420 may be substituted, for example, with glutamic acid (K420E), aspartic acid (K420D), leucine (K420L) or methionine (K410M); Asparagine N527 may be substituted, for example, with glutamine (N527Q), aspartic acid (N527D), histidine (N527H), or tyrosine (N527Y); Glutamic acid E505 may be substituted, for example, with glycine G (E505G), alanine (E505A), leucine (E505L), lysine (E505K), valine (E505V), isoleucine (E505I), methionine (E505M), or glutamine (E505Q); Valine V547 may be substituted with, for example, alanine (V547A), glycine (V547G), leucine (V547L), lysine (V547K), isoleucine (V547I), methionine (V547M); Asparagine N503 and / or N527 may be substituted with, for example, glutamine (N503Q and / or N527Q), aspartic acid (N503D and / or N527D), histidine (N503H and / or N527H), or tyrosine (N503Y and / or N527Y).

[0144] In some embodiments, the HSA variant (referred to herein as mHSA) has the following substitutions: C34S, N503Q (SEQ ID NO: 13). In some embodiments, the HSA variant (referred to herein as mHSA7) has the following substitutions: C34S, N503Q, E505G, and V547A (SEQ ID NO: 14). In some embodiments, the HSA variant has amino acids 26-609 and the following substitutions: C58S and N527Q (SEQ ID NO: 15).

[0145] In some embodiments, asparagine at positions 503 and / or 527 of HSA, which may be deamidated and reduce half-life, may be removed by N503Q substitution and / or N527Q substitution. In some embodiments, cysteine ​​C34 of HSA may be substituted with serine or alanine (S or A) to remove the free cysteine ​​and minimize alternative disulfide bond formation. In some embodiments, the half-life modulator is a modified version of domain III of modified HSA (mHSA_dIII) with an N503Q substitution and an additional terminal glycine. Such a modified version maintains the properties of HSA, such as binding to FcRn and increased serum half-life.

[0146] In some embodiments, the half-life modulator is an antibody Fc domain or single-chain constant fragment. In some embodiments, the half-life modulator comprises an Fc region of an immunoglobulin molecule (e.g., IgG). In some embodiments, the half-life modulator comprises at least 100 contiguous amino acids that are at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a human Fc amino acid sequence. The antibody Fc domain has the natural ability to bind to FcRn, resulting in extended half-life. In some embodiments, the antibody Fc domain is engineered to not bind to Fc(gamma)R. In an exemplary embodiment, the Fc domain is engineered to replace N297 with Q (N297Q variant). In some embodiments, the half-life modulator is a monomeric variant form of Fc (scFc). For example, a subset of IgG heavy chains that naturally dimerize to form Fc are hinge-CH2-CH3. In some embodiments, the Fc domain is engineered to form a single chain by joining hinge-CH2-CH3 with a flexible linker, such as GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 16), creating a hinge-CH2-CH3-linker-hinge-CH2-CH3 chain. In an exemplary embodiment, a single-chain Fc (scFc) is engineered to replace N297 with Q and C220 with S (N297Q, C220S).

[0147] In some embodiments, the half-life modulator is a single-chain variable fragment (scFv) of an antibody that targets albumin or other circulating protein. In some embodiments, the half-life modulator comprises an amino acid sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of an scFv against a particular antigen, such as, but not limited to, albumin. In some embodiments, the half-life modulator comprises at least 50, at least 100, at least 150, at least 200, or at least 250 contiguous amino acids that are at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of an scFv against a particular antigen, such as, but not limited to, albumin.

[0148] In some embodiments, the half-life modulator is transferrin, such as human transferrin (Tf, SEQ ID NO: 17). In some embodiments, the half-life modulator comprises an amino acid sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a human transferrin amino acid sequence. In some embodiments, the half-life modulator comprises at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, or at least 650 contiguous amino acids that are at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a human transferrin amino acid sequence.

[0149] In some embodiments, the half-life modulator comprises at least 100 contiguous amino acids that are at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the wild-type human alpha-fetoprotein amino acid sequence (AFP, SEQ ID NO: 18). In some embodiments, the half-life modulator comprises at least 100 contiguous amino acids that are about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the wild-type human alpha-fetoprotein (AFP) amino acid sequence. In some embodiments, the N-linked glycosylation site of AFP is eliminated by a N251Q substitution.

[0150] In some embodiments, the half-life modulator comprises at least 100 contiguous amino acids that are at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the wild-type vitamin D binding protein amino acid sequence (VDBP, SEQ ID NO: 19). In some embodiments, the half-life modulator comprises at least 100 contiguous amino acids that are about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the wild-type vitamin D binding protein (VDBP) amino acid sequence. In some embodiments, the N-linked glycosylation site of VDBP can be eliminated by an N288Q or N288T substitution.

[0151] In some embodiments, the half-life modulator comprises at least 100 contiguous amino acids that are at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the wild-type human transthyretin amino acid sequence (TTR, SEQ ID NO: 20). In some embodiments, the half-life modulator comprises at least 100 contiguous amino acids that are about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the wild-type human transthyretin (TTR) amino acid sequence. In some embodiments, the transthyretin is modified to remove the N118 N-glycosylation site. In some embodiments, the half-life modulator is a monomeric form of TTR.

[0152] In some embodiments, the half-life modulator comprises at least 100 contiguous amino acids that are at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a PASylated amino acid sequence. The PASylated amino acid sequence is a proline-, alanine-, and / or serine-rich sequence that mimics PEGylation (see WO / 2008 / 155134). In some embodiments, the half-life modulator comprises at least 100 contiguous amino acids that are about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a PASylated amino acid sequence. The PASylated amino acid sequence is a proline-, alanine-, and / or serine-rich sequence that mimics PEGylation. The polypeptide stretch of proline, alanine, and / or serine forms a semi-structured three-dimensional domain with a large hydrodynamic radius, thereby reducing the clearance of the fusion protein. In some embodiments, the PAS-modified amino acid sequence is about 200, 300, 400, 500, or 600 amino acids long. For example, the PAS-modified amino acid sequence is ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 21), which is a 20-time repeat of the amino acid sequence ASPAAPAPASPAAPAPSAPA.

[0153] In some embodiments, the half-life modulator comprises a polyethylene glycol (PEG) chain or chains attached to the fusion protein via chemical attachment to either the N- and / or C-terminus and / or amino acid side chain (e.g., PEG-maleimide attachment to cysteine). The PEG chains form semi-structured three-dimensional domains with large hydrodynamic radii, thereby reducing clearance of the fusion protein.

[0154] In some embodiments, the half-life modulator comprises at least 100 contiguous amino acids that are at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an albumin binding domain human antibody (albudAb) amino acid sequence (SEQ ID NO: 22). In some embodiments, the half-life modulator comprises at least 100 contiguous amino acids that are about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an albumin binding domain human antibody (albudAb) amino acid sequence. Albumin binding domain antibodies may increase the half-life of the fusion protein by non-covalently binding to serum albumin (see WO2008 / 096158, incorporated herein by reference in its entirety). In some embodiments, the albumin binding domain human antibody is engineered to remove the C-terminal arginine and the Lys-ArgKex2 protease site.

[0155] Representative such half-life modulators include those set forth in any one of SEQ ID NOs: 12-15, 17-22.

[0156] In some embodiments, half-life modulators can be modified to replace cysteine ​​residues with serine or alanine residues, reducing the ability to form disulfides.

[0157] In some embodiments, the targeting domain and the activator domain can be linked via a half-life modulator. Thus, the half-life modulator can have two termini, an N-terminus and a C-terminus. In some embodiments, the half-life modulator is linked to the targeting polypeptide domain at one end via a peptide bond and to the activator domain at the other end via a peptide bond. In certain embodiments, the half-life modulator is linked N-terminally to the C-terminus of the targeting polypeptide domain and C-terminally to the N-terminus of the activator domain. In other embodiments, the half-life modulator is linked C-terminally to the targeting polypeptide domain and N-terminally to the activator domain. However, in other embodiments, the half-life modulator is linked to one of the termini of the bispecific protein. For example, in some embodiments, the half-life modulator is linked C-terminally to the N-terminus of the activator domain. In other embodiments, the half-life modulator is linked N-terminally to the C-terminus of the targeting domain. In other embodiments, the half-life modulator can be linked N-terminally to the C-terminus of the activator domain, however, in other embodiments, the half-life modulator can be linked N-terminally to the C-terminus of the targeting domain.

[0158] Peptide Linker In some embodiments, the activator domain, half-life modulator, and targeting domain are linked by a peptide linker (e.g., 2 to 40, 2-50, 2-100 amino acid residues), such that upon target recognition and engagement by the targeting domain, the presentation of the activator domain is at a predetermined surface density (e.g., −5×10 2 molecules / 1,000 Å2) and are optimized for binding to and activation of extracellular receptors on the surface of target cells.

[0159] Targeted delivery of an activator domain, such as IGF-1, to activate a receptor on a cell or tissue that presents a specific target requires the proper presentation of both the activator domain and the targeting domain. In some embodiments, the flexibility of the linker is optimized to ensure the proper geometric structure of the linked chimeric protein. The primary determinant of some geometric constraints is the distance from the cell surface to the target and receptor.

[0160] Additional optimization can be driven by the relative numbers of receptor and target molecules. When the receptor:target molecule ratio is large, binding of both domains is reaction-limited. When target molecules are more abundant than receptors, occupancy of both domains is diffusion-limited. Under reaction-limited conditions, optimal delivery of the activator domain is achieved via a short, rigid linker. Under diffusion-limited conditions, a long, flexible linker allows the activator domain to access a large surface area. For cells with complex geometries (i.e., somatic and neural processes) and receptor distribution, appropriate design of linker flexibility can enable precise targeting to subcellular regions.

[0161] In some embodiments, the peptide linker is present at one or both ends of the half-life modulator, at the N-terminus, the C-terminus, or both the N-terminus and the C-terminus. Short connector polypeptides suitable for use at the N-terminus of the linker include dipeptides such as -Gly-Ser-(GS), -Gly-Ala-(GA), and -Ala-Ser-(AS). Such peptides suitable for use at the C-terminus of the linker include dipeptides such as -Leu-Gln-(LQ) and -Thr-Gly-(TG). In some embodiments, the peptide linker is longer than two amino acids. For example, the peptide linker is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length or longer. In some embodiments, the peptide linker is 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more amino acids in length. Preferably, such peptide linkers are flexible (e.g., glycine-rich) or structured (e.g., alpha-helix-rich). In some embodiments, the linker comprises or consists of the amino acids -Gly-Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 23).

[0162] It will be apparent that elements in addition to those described above may optionally be included in the proteins provided herein. Such elements may be present for a variety of purposes, including facilitating expression, preparing or purifying bispecific fusion proteins, or performing a targeting function.

[0163] Representative chimeric proteins In some embodiments, an exemplary bispecific fusion protein comprises (N-terminus to C-terminus): (a) a targeting polypeptide domain comprising or consisting of a non-internalizing human annexin V variant (e.g., comprising or consisting of amino acids 2-320 of wt human annexin V and substitutions at C316, R63, K40, K101, E138, D139, N160); (b) a linker peptide (e.g., -Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly); (c) half-life modulators (e.g., HSA variants comprising or consisting of amino acids 26-609 of wt human HSA and containing substitutions at C58 and N527); (d) linker peptides (e.g., -Gly-Ser-Gly-Gly-Gly-Ser-Gly); (e) an activator domain comprising or consisting of an IGF-1 variant (e.g., comprising substitutions at E3 and Y31). Includes.

[0164] In some embodiments, the chimeric protein comprises or consists of IGF1(E3R / Y31A)_lk7_HSA26-609(C58S / N527Q)_lk7_AnxV2-320(R63A / K70A / K101A / E138A / D139G / N160A / C316A) (also referred to herein as scp776). In some embodiments, the chimeric protein has the amino acid sequence set forth in SEQ ID NO:24.

[0165] In some embodiments, the chimeric protein comprises or consists of IGF1(E3R / Y31A)_lk7_HSA26-609(C58S / N527Q)_lk7_AnxV2-320(R63A / K70A / K101A / E138A / D139G / N160A / C316S). In some embodiments, the chimeric protein has the amino acid sequence set forth in SEQ ID NO:25.

[0166] nucleic acid The polynucleotides provided herein encode chimeric proteins, and can be in the form of RNA or DNA, including cDNA and synthetic DNA.DNA can be double-stranded or single-stranded.The coding sequences encoding variants of the present disclosure can be different due to redundancy or degeneracy of genetic code.

[0167] Pharmaceutical Compositions - Methods of Treatment Pharmaceutical compositions are provided that comprise a therapeutically effective amount of at least one chimeric protein described herein in association with at least one physiologically acceptable carrier. Such compositions may be used to prevent tissue damage or to repair or regenerate damaged tissue in patients undergoing treatment who have experienced or are at risk of tissue damage.

[0168] In some embodiments, the subject in need thereof has an acute CNS disorder, an acute cardiovascular disorder (e.g., STEMI, cardiac arrest), acute radiation sickness (e.g., acute radiation syndrome (ARS) of the gastrointestinal (GI-ARS) and hematopoietic system (H-ARS)), a chemical inhalation injury (e.g., sulfur mustard exposure), a dermatological disorder (e.g., wound healing), a traumatic injury (e.g., burns, crush injuries, lacerations, contusions, avulsions, concussions, fractures, amputations), or a chronic neurodegenerative disease (e.g., synucleinopathy, amyloidosis). In some embodiments, the subject in need thereof is undergoing a procedure (e.g., transcatheter aortic valve replacement, percutaneous coronary intervention, coronary artery bypass graft surgery) that carries a risk of iatrogenic injury. In some embodiments, the subject in need thereof is undergoing an organ transplant procedure (e.g., skin, kidney, liver, heart, lung). In some embodiments, the subject is undergoing a cosmetic dermatological treatment (e.g., laser resurfacing).

[0169] In some embodiments, the pharmaceutical composition is a liquid formulation prepared for intravenous (IV) injection, hi some embodiments, the composition is prepared for IV bolus administration.

[0170] In some embodiments, the pH of the pharmaceutical composition is about 7 to about 8, e.g., about 7.5. In some embodiments, the pharmaceutical composition includes an alkalizing agent, such as tromethamine or dibasic sodium phosphate. In some embodiments, alkalinization is achieved at a concentration of about 10 to about 50 mM, e.g., about 20 mM. In some embodiments, the pharmaceutical composition includes a surfactant. In some embodiments, the surfactant is a non-ionic surfactant, such as polysorbate 80 or polysorbate 20, and may be present at about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (w / v), or any concentration therebetween. In some embodiments, the pharmaceutical composition includes sucrose. In some embodiments, sucrose may be present at a concentration of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% (w / v) or therebetween.

[0171] In some embodiments, the pharmaceutical composition comprises about 1 to about 100 g / L chimeric protein, about 10 mM to about 50 mM tromethamine, about 2 to about 15% (w / v) sucrose, about 0.001% to about 0.04% (w / v) polysorbate 80 at pH 7.5.

[0172] In some embodiments, the chimeric protein (about 1 to about 100 g / L) is prepared in about 20 mM tromethamine, about 7.5% sucrose, and about 0.02% polysorbate 80, pH 7.5 (by adding HCl). In some embodiments, about 20 mg / ml of the chimeric protein is prepared in about 20 mM tromethamine, about 7.5% sucrose, and about 0.02% polysorbate 80, pH 7.5 (by adding HCl).

[0173] In some embodiments, the pH of the pharmaceutical composition is about 4.5 to about 6.5, about 4.5 to about 5, about 5 to about 5.5, about 5.5 to about 6, about 6 to about 6.5, about 4.5, about 5, about 5.5, about 6, or about 6.6. In some embodiments, the pharmaceutical composition comprises acetate, phosphate, citrate, or histidine, e.g., at a concentration of 10 to 50 mM. In some embodiments, the pharmaceutical composition comprises acetate. In some embodiments, the citrate may be at a concentration of about 10 to about 50 mM, e.g., about 20 mM. In some embodiments, the pharmaceutical composition comprises sucrose. In some embodiments, the sucrose may be present at a concentration (w / v) of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or therebetween.

[0174] In some embodiments, the pharmaceutical composition comprising the chimeric protein comprises citrate, sucrose, or a combination thereof.

[0175] In some embodiments, the pharmaceutical composition comprises less than 2% (w / v) sucrose, less than 1.5% (w / v) sucrose, less than 1% (w / v) sucrose, less than 0.5% (w / v) sucrose, less than 0.1% (w / v) sucrose, or less than 0.01% (w / v) sucrose. In some embodiments, the pharmaceutical composition is sucrose-free.

[0176] In some embodiments, the pharmaceutical composition comprises about 1 to about 25 g / l chimeric protein, or any value therebetween.

[0177] In some embodiments, the pharmaceutical composition has a pH of about 5 to about 7, e.g., about 5, about 5.5, about 6, about 6.5, about 7.

[0178] In some embodiments, the pharmaceutical composition comprises about 100 mM to about 150 mM sodium chloride and sterile water. In some embodiments, the pharmaceutical composition is a preservative-free formulation. In some embodiments, the pharmaceutical composition is sterile.

[0179] In some embodiments, the pharmaceutical composition comprises an excipient such as, but not limited to, histidine, histidine monohydrochloride, histidine hydrochloride, methionine, succinic acid, trehalose dihydrate, polysorbate 20, and polysorbate 80.

[0180] In some embodiments, the pharmaceutical composition has an osmolality of about 45-140 mOsmol / kg, or any value therebetween, for example, 45-75 mOsmol / kg, 74-110 mOsmol / kg, or 110-140 mOsmol / kg.

[0181] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the chimeric protein, about 20 mM sodium citrate, about 122 mM sodium chloride, and has a pH of about 6.5. In some embodiments, the pharmaceutical composition consists essentially of a therapeutically effective amount of the chimeric protein, about 20 mM sodium citrate, about 122 mM sodium chloride, and has a pH of about 6.5. In some embodiments, the pharmaceutical composition consists of a therapeutically effective amount of the chimeric protein, about 20 mM sodium citrate, about 122 mM sodium chloride, and has a pH of about 6.5.

[0182] In some embodiments, the pharmaceutical composition comprises about 1 to about 25 g / L chimeric protein, about 2 to about 15% (w / v) sucrose at pH 5.

[0183] In some embodiments, the chimeric protein (about 1 to about 10 g / L) is prepared in about 20 mM citrate, about 7.5% sucrose, pH 5. In some embodiments, about 5 mg / ml of the chimeric protein is prepared in about 20 mM citrate, about 7.5% sucrose, pH 5.

[0184] In some embodiments, the pharmaceutical composition comprising the chimeric protein comprises an alkalizing agent, a surfactant, sucrose, or a combination thereof.

[0185] The pharmaceutical compositions of the present disclosure may contain pharmaceutically acceptable auxiliary substances required to achieve a near-physiological state, such as pH adjusting and buffering substances, osmotic pressure adjusting substances, wetting substances, detergents, antiseptic substances, and the like. In some embodiments, the liquid composition is sterilized by conventional sterilization techniques or sterile filtered. In some embodiments, the liquid composition is present in a vial.

[0186] In some embodiments, the pharmaceutical composition is administered intravenously or intraarterially to a subject in need thereof via a bolus administration. Bolus administration includes, for example, a fast intravenous injection in less than 10 seconds (or less than 20, 30, 40, 50, or 60 seconds), or an intravenous infusion in less than approximately 3, 4, 5, 6, 7, 8, 9, or 10 minutes. In some embodiments, the pharmaceutical composition is administered intravenously to a subject in need thereof. In some embodiments, the composition is administered by IV push. In some embodiments, administration occurs in 5 minutes or less, 4 minutes or less, 3 minutes or less, 2 minutes or less, or 1 minute or less (e.g., 50 seconds, 40 seconds, 30 seconds, 20 seconds, or any administration time therebetween). In other embodiments, the composition is administered by slow IV injection.

[0187] In some embodiments, the pharmaceutical composition is intravenously administered to a subject using a syringe injection pump at a rate of 0.5-25mL / min. For example, the pharmaceutical composition is intravenously administered to a subject using a syringe injection pump at a rate of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5 or 25mL / min. In some embodiments, the pharmaceutical composition is administered using a syringe injection pump in doses of 0.5-1, 1-1.5, 1.5-2, 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, 6.5-7, 7-7.5, 7.5-8, 8-8.5, 8.5-9, 9-9.5, 9.5-10, 10-10.5, 10.5-11, 11-11.5, 11.5-12, 12-12.5, 12.5-13, 13-13.5, 1 It is administered intravenously to the subject at a rate of 3.5-14, 14-14.5, 14.5-15, 15-15.5, 15.5-16, 16-16.5, 16.5-17, 17-17.5, 17.5-18, 18-18.5, 18.5-19, 19-19.5, 19.5-20, 20-20.5, 20.5-21, 21-21.5, 21.5-22, 22-22.5, 22.5-23, 23-23.5, 23.5-24, 24-24.5, or 24.5-25 mL / min.

[0188] In some embodiments, the pharmaceutical composition is administered intra-arterially to a subject using a syringe injection pump at a rate of 0.05-10 mL / min. For example, the pharmaceutical composition is administered intra-arterially to a subject using a syringe injection pump at a rate of 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mL / min. In some embodiments, the pharmaceutical composition is administered intra-arterially to a subject using a syringe injection pump at a rate of 0.05-0.1, 0.1-0.2, 0.2-0.3, 0.3-0.4, 0.4-0.5, 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, 0.9-1, 1-1.5, 1.5-2, 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, 6.5-7, 7-7.5, 7.5-8, 8-8.5, 8.5-9, 9-9.5, 9.5-10 mL / min.

[0189] In some embodiments, the pharmaceutical composition is administered to the subject intrathecally using techniques known in the art.

[0190] In some embodiments, a therapeutically effective amount generally ranges from about 0.01 mg / kg to about 100.0 mg / kg per administration.In some embodiments, a therapeutically effective amount of a protein disclosed herein is from about 0.01 mg / kg to about 0.05 mg / kg per dose, from about 0.01 mg / kg to about 0.1 mg / kg per dose, from 0.01 mg / kg to about 1 mg / kg per dose, from 0.01 mg / kg to about 10 mg / kg per dose, from 0.01 mg / kg to about 100.0 mg / kg per dose, from 0.1 mg / kg to about 0.5 mg / kg per dose, from 0.1 mg / kg to about 1 mg / kg per dose, from 0.1 mg / kg to about 5 mg / kg per dose, / kg, 0.1mg / kg to about 10mg / kg per dose, 0.1mg / kg to about 20mg / kg per dose, 0.1mg / kg to about 30mg / kg per dose, 0.1mg / kg to about 40mg / kg per dose, 0.1mg / kg to about 50mg / kg per dose, 0.1mg / kg to about 60mg / kg per dose, 0.1mg / kg to about 70mg / kg per dose, 0.1mg / kg to about 80mg / kg per dose, 0.1mg / kg to about 90mg / kg per dose, 0.1mg / kg to about 100mg / kg per dose, 0.1mg / kg to about 150mg / kg per dose, 0.1mg / kg to about 200mg / kg per dose, 0.1mg / kg to about 300mg / kg per dose, 0.1mg / kg to about 40mg / kg per dose, 0.1mg / kg to about 50mg / kg per dose, 0.1mg / kg to about 60mg / kg per dose, 0.1mg / kg to about 70mg / kg per dose, 0.1mg / kg to about 80mg / kg per dose, 0.1mg / kg to about 90mg / kg per dose, 0.1mg / kg to about 150mg / kg per dose, about 100 mg / kg, 1 mg / kg to about 5 mg / kg per dose, 1 mg / kg to about 10 mg / kg per dose, 1 mg / kg to about 20 mg / kg per dose, 1 mg / kg to about 30 mg / kg per dose, 1 mg / kg to about 40 mg / kg per dose, 1 mg / kg to about 50 mg / kg per dose, 1 mg / kg to about 60 mg / kg per dose, 1 mg / kg to about 70 mg / kg per dose, 1 mg / kg to about 80 mg / kg per dose, 1 mg / kg to about 90 mg / kg per dose, and 10 mg / kg to about 100 mg / kg per dose, 10 mg / kg to about 20 mg / kg per dose, 10 mg / kg to about 30 mg / kg per dose, 10 mg / kg to about 40 mg / kg per dose, 10 mg / kg to about 50 mg / kg per dose, 10 mg / kg to about 60 mg / kg per dose, 10 mg / kg to about 70 mg / kg per dose, 10 mg / kg to about 80 mg / kg per dose, 10 mg / kg to about 90 mg / kg per dose, or 10 mg / kg to about 100 mg / kg per dose.

[0191] In some embodiments, a therapeutically effective amount generally ranges from about 0.01 mg / kg to about 20.0 mg / kg per dose, hi some embodiments, a therapeutically effective amount ranges from about 0.01 mg / kg to about 10.0 mg / kg per dose.

[0192] In some embodiments, a therapeutically effective amount of a protein disclosed herein is from about 0.01 mg / kg to about 0.02 mg / kg per dose, from about 0.01 mg / kg to about 0.03 mg / kg per dose, from about 0.01 mg / kg to about 0.04 mg / kg per dose, from about 0.01 mg / kg to about 0.05 mg / kg per dose, from about 0.01 mg / kg to about 0.06 mg / kg per dose, from about 0.01 mg / kg to about 0.07 mg / kg per dose, from about 0.01 mg / kg to about 0.08 mg / kg per dose, from about 0.01 mg / kg to about 0.09 mg / kg per dose, from about 0.01 mg / kg to about 0.10 mg / kg per dose, from about 0.01 mg / kg to about 0.11 mg / kg per dose, from about 0.01 mg / kg to about 0.12 mg / kg per dose, from about 0.01 mg / kg to about 0.13 mg / kg per dose, from about 0.01 mg / kg to about 0.14 mg / kg per dose, from about 0.01 mg / kg to about 0.15 mg / kg per dose, from about 0.01 mg / kg to about 0.16 mg / kg per dose, from about 0.01 mg / kg to about 0.17 mg / kg per dose, from about 0.01 mg / kg to about 0.18 mg / kg per dose, from about 0.01 mg / kg to about 0.19 mg / kg per dose, from about 0.01 mg / kg to about 0.20 mg / kg per dose, from about 0.01 mg / kg to about 0.21 mg / kg per dose, from about 0.01 mg / kg to about 0.22 mg / kg per dose, from about 0.01 mg / kg to about about 0.01 mg / kg to about 0.09 mg / kg per dose, about 0.01 mg / kg to about 0.1 mg / kg per dose, about 0.01 mg / kg to about 0.2 mg / kg per dose, about 0.01 mg / kg to about 0.3 mg / kg per dose, about 0.01 mg / kg to about 0.4 mg / kg per dose, about 0.01 mg / kg to about 0.5 mg / kg per dose, about 0.01 mg / kg to about 0.6 mg / kg per dose, about 0.01 mg / kg to about 0.7 mg / kg per dose, about 0.01 mg / kg to about 0.8 mg / kg per dose, about 0.01 mg / kg to about 0.9 mg / kg per dose, about 0.01 mg / kg to about 0.1 mg / kg per dose, about 0.01 mg / kg to about 0.2 mg / kg per dose, about 0.01 mg / kg to about 0.3 mg / kg per dose, about 0.01 mg / kg to about 0.4 mg / kg per dose, about 0.01 mg / kg to about 0.5 mg / kg per dose, about 0.01 mg / kg to about 0.6 mg / kg per dose, about 0.01 mg / kg to about 0.7 mg / kg per dose, about 0.01 mg / kg to about 0.8 mg / kg per dose kg, about 0.01 mg / kg to about 0.9 mg / kg per dose, about 0.01 mg / kg to about 1 mg / kg per dose, about 0.01 mg / kg to about 2 mg / kg per dose, about 0.01 mg / kg to about 3 mg / kg per dose, about 0.01 mg / kg to about 4 mg / kg per dose, about 0.01 mg / kg to about 5 mg / kg per dose, about 0.01 mg / kg to about 6 mg / kg per dose, about 0.01 mg / kg to about 7 mg / kg per dose, about 0.01 mg / kg to about 8 mg / kg per dose, About 0.01 mg / kg to about 9 mg / kg, about 0.01 mg / kg to about 10 mg / kg per dose, about 0.01 mg / kg to about 11 mg / kg per dose, about 0.01 mg / kg to about 12 mg / kg per dose, about 0.01 mg / kg to about 13 mg / kg per dose, about 0.01 mg / kg to about 14 mg / kg per dose, about 0.01 mg / kg to about 15 mg / kg per dose, about 0.01 mg / kg to about 16 mg / kg per dose, about 0.01 mg / kg to about 17 mg / kg per dose, about 0.The ranges are from about 0.01 mg / kg to about 18 mg / kg per dose, from about 0.01 mg / kg to about 19 mg / kg per dose, and from about 0.01 mg / kg to about 20 mg / kg per dose.

[0193] In some embodiments, a therapeutically effective amount of a protein disclosed herein is from about 0.01 mg / kg to about 0.05 mg / kg per dose, from about 0.05 mg / kg to about 0.1 mg / kg per dose, from about 0.1 mg / kg to about 0.5 mg / kg per dose, from about 0.05 mg / kg to about 1 mg / kg per dose, from about 1 mg / kg to about 2 mg / kg per dose, from about 2 mg / kg to about 3 mg / kg per dose, from about 3 mg / kg to about 4 mg / kg per dose, from about 4 mg / kg to about 5 mg / kg per dose, from about 5 mg / kg to about 6 mg / kg per dose, from about 6 mg / kg to about 7 mg / kg per dose, from about 7 mg / kg to about 8 mg / kg per dose, The ranges are: about 8 mg / kg to about 9 mg / kg per dose, about 9 mg / kg to about 10 mg / kg per dose, about 10 mg / kg to about 11 mg / kg per dose, about 11 mg / kg to about 12 mg / kg per dose, about 12 mg / kg to about 13 mg / kg per dose, about 13 mg / kg to about 14 mg / kg per dose, about 14 mg / kg to about 15 mg / kg per dose, about 15 mg / kg to about 16 mg / kg per dose, about 16 mg / kg to about 17 mg / kg per dose, 17 mg / kg to about 18 mg / kg per dose, about 18 mg / kg to about 19 mg / kg per dose, and about 19 mg / kg to about 20 mg / kg per dose.

[0194] In some embodiments, a therapeutically effective amount of a protein disclosed herein is about 0.01 mg / kg per dose, about 0.02 mg / kg per dose, about 0.03 mg / kg per dose, about 0.04 mg / kg per dose, about 0.05 mg / kg per dose, about 0.06 mg / kg per dose, about 0.07 mg / kg per dose, about 0.08 mg / kg per dose, about 0.09 mg / kg per dose, about 0.1 mg / kg per dose, about 0.2 mg / kg per dose, about 0.3 mg / kg per dose, about 0. 4mg / kg, about 0.5mg / kg per dose, about 0.6mg / kg per dose, about 0.7mg / kg per dose, about 0.8mg / kg per dose, about 0.9mg / kg per dose, about 1mg / kg per dose, about 1.1mg / kg per dose, about 1.2mg / kg per dose, about 1.3mg / kg per dose, about 1.4mg / kg per dose, about 1.5mg / kg per dose, about 1.6mg / kg per dose, about 1.7mg / kg per dose, about 1.8mg / kg per dose, about 1.9mg / kg per dose, about 2mg / kg per dose, about 2.1mg / kg per dose, about 2.2mg / kg per dose, about 2.3mg / kg per dose, about 2.4mg / kg per dose, about 2.5mg / kg per dose, about 2.6mg / kg per dose, about 2.7mg / kg per dose, about 2.8mg / kg per dose, about 2.9mg / kg per dose, about 3mg / kg per dose, about 3.1mg / kg per dose, about 3.2mg / kg per dose, about 3.3mg / kg per dose, about 3.4mg / kg per dose, about 3.5mg / kg per dose kg, about 3.6mg / kg per dose, about 3.7mg / kg per dose, about 3.8mg / kg per dose, about 3.9mg / kg per dose, about 4mg / kg per dose, about 4.1mg / kg per dose, about 4.2mg / kg per dose, about 4.3mg / kg per dose, about 4.4mg / kg per dose, about 4.5mg / kg per dose, about 4.6mg / kg per dose, about 4.7mg / kg per dose, about 4.8mg / kg per dose, about 4.9mg / kg per dose, about 5mg / kg per dose, about 5.1mg / kg, about 5.2mg / kg per dose, about 5.3mg / kg per dose, about 5.4mg / kg per dose, about 5.5mg / kg per dose, about 5.6mg / kg per dose, about 5.7mg / kg per dose, about 5.8mg / kg per dose, about 5.9mg / kg per dose, about 6mg / kg per dose, about 6.1mg / kg per dose, about 6.2mg / kg per dose, about 6.3mg / kg per dose, about 6.4mg / kg per dose, about 6.5mg / kg per dose, about 6.6mg / kg per dose, about 6.7mg / kg per dose, about 6.8mg / kg per dose, about 6.9 ... about 6.6mg / kg per dose, about 6.7mg / kg per dose, about 6.8mg / kg per dose, about 6.9mg / kg per dose, about 7mg / kg per dose, about 7.1mg / kg per dose, about 7.2mg / kg per dose, about 7.3mg / kg per dose, about 7.4mg / kg per dose, about 7.5mg / kg per dose, about 7.6mg / kg per dose, about 7.7mg / kg per dose, about 7.8mg / kg per dose, about 7.9mg / kg per dose, about 8mg / kg per dose, about 8.1 mg / kg per dose, about 8.2 mg / kg per dose, about 8.3 mg / kg per dose, about 8.4 mg / kg per dose, about 8.5 mg / kg per dose, about 8.6 mg / kg per dose, about 8.7 mg / kg per dose, about 8.8 mg / kg per dose, about 8.9 mg / kg per dose, about 9 mg / kg per dose, about 9.1 mg / kg per dose, about 9.2 mg / kg per dose, about 9.3 mg / kg per dose, about 9.4 mg / kg per dose, about 9.5 mg / kg per dose g, about 9.6 mg / kg per dose, about 9.7 mg / kg per dose, about 9.8 mg / kg per dose, about 9.9 mg / kg per dose, about 10 mg / kg per dose, about 11 mg / kg per dose, about 12 mg / kg per dose, about 13 mg / kg per dose, about 14 mg / kg per dose, about 15 mg / kg per dose, about 16 mg / kg per dose, about 17 mg / kg per dose, about 18 mg / kg per dose, about 19 mg / kg per dose, and about 20 mg / kg per dose.

[0195] In some embodiments, a therapeutically effective amount is generally in the range of about 1 mg / kg to about 10.0 mg / kg per dose. In some embodiments, an effective amount of a protein disclosed herein can be, for example, about 1 mg / kg to about 10 mg / kg per dose, 1 mg / kg to about 9 mg / kg per dose, 1 mg / kg to about 8 mg / kg per dose, 1 mg / kg to about 7 mg / kg per dose, 1 mg / kg to about 6 mg / kg per dose, 1 mg / kg to about 5 mg / kg per dose, 1 mg / kg to about 4 mg / kg per dose, 1 mg / kg to about 3 mg / kg per dose, or 1 mg / kg to about 2 mg / kg per dose.

[0196] Administration can be single or cumulative (continuous), and can be easily determined by one skilled in the art. For example, treatment of a nervous system disorder can include a single administration of an effective dose of the pharmaceutical composition disclosed herein. As a non-limiting example, an effective dose of the composition disclosed herein can be administered to a patient once, for example, as a single injection or bolus. Alternatively, treatment of a nervous system disorder can include multiple administrations of an effective dose of the pharmaceutical composition disclosed herein, administered over a range of time periods, such as four times a day, three times a day, twice a day, once a day, once every few days, once a week, once a month, or once a year. As a non-limiting example, the combination disclosed herein can be administered to a patient once or twice a week. The timing of administration can depend on factors such as the severity of the patient's symptoms. For example, an effective dose of the composition disclosed herein can be administered to a patient once a month indefinitely, or until the mammal no longer requires treatment.

[0197] In some embodiments, a therapeutically effective amount generally ranges from about 0.01 mg / kg to about 200.0 mg / kg per day. In some embodiments, an effective amount of a protein disclosed herein ranges from about 0.01 mg / kg to about 0.1 mg / kg per day, 0.01 mg / kg to about 1 mg / kg per day, 0.01 mg / kg to about 10 mg / kg per day, 0.01 mg / kg to about 100.0 mg / kg per day, 0.01 mg / kg to about 200.0 mg / kg per day, 0.1 mg / kg to about 1 mg / kg per day, 0.1 mg / kg to about 10 mg / kg per day, 0.1 mg / kg to about 100 mg / kg per day, 0.1 mg / kg to about 200 mg / kg per day, 1 mg / kg to about 100 mg / kg per day, 1 mg / kg to about 200 mg / kg per day, 10 mg / kg to about 100 mg / kg per day, or 10 mg / kg to about 100 mg / kg per day.

[0198] In some embodiments, a therapeutically effective amount is generally within the range of about 0.01 mg / kg to about 20.0 mg / kg per day. In some embodiments, a therapeutically effective amount is generally within the range of about 0.01 mg / kg to about 10.0 mg / kg per day. In some embodiments, a therapeutically effective amount is generally within the range of about 1 mg / kg to about 20.0 mg / kg per day. In some embodiments, a therapeutically effective amount is generally within the range of about 1 mg / kg to about 10.0 mg / kg per day. In some embodiments, an effective amount of a protein disclosed herein can be, for example, from about 1 mg / kg to about 10 mg / kg per day, from 1 mg / kg to about 9 mg / kg per day, from 1 mg / kg to about 8 mg / kg per day, from 1 mg / kg to about 7 mg / kg per day, from 1 mg / kg to about 6 mg / kg per day, from 1 mg / kg to about 5 mg / kg per day, from 1 mg / kg to about 4 mg / kg per day, from 1 mg / kg to about 3 mg / kg per day, or from 1 mg / kg to about 2 mg / kg per day.

[0199] In some embodiments, a therapeutically effective amount of a chimeric protein disclosed herein is from about 0.01 mg / kg to about 0.02 mg / kg per day, from about 0.01 mg / kg to about 0.03 mg / kg per day, from about 0.01 mg / kg to about 0.04 mg / kg per day, from about 0.01 mg / kg to about 0.05 mg / kg per day, from about 0.01 mg / kg to about 0.06 mg / kg per day, from about 0.01 mg / kg to about 0.07 mg / kg per day, from about 0.01 mg / kg to about 0.08 mg / kg per day, from about 0.01 mg / kg to about 0.09 mg / kg per day, from about 0.01 mg / kg to about 0.10 mg / kg per day, from about 0.01 mg / kg to about 0.11 mg / kg per day, from about 0.01 mg / kg to about 0.12 mg / kg per day, from about 0.01 mg / kg to about 0.13 mg / kg per day, from about 0.01 mg / kg to about 0.14 mg / kg per day, from about 0.01 mg / kg to about 0.15 mg / kg per day, from about 0.01 mg / kg to about 0.16 mg / kg per day, from about 0.01 mg / kg to about 0.17 mg / kg per day, from about 0.01 mg / kg to about 0.18 mg / kg per day, from about 0.01 mg / kg to about 0.19 mg / kg per day, from about 0.01 mg / kg to about 0.20 mg / kg per day, from about 0.01 mg / kg to about 0.21 mg / kg per day, from about 0.01 mg / kg to about 0.22 mg / kg per day, from about 0.01 mg / kg to 0.01mg / kg to about 0.09mg / kg, about 0.01mg / kg to about 0.1mg / kg per day, about 0.01mg / kg to about 0.2mg / kg per day, about 0.01mg / kg to about 0.3mg / kg per day, about 0.01mg / kg to about 0.4mg / kg per day, about 0.01mg / kg to about 0.5mg / kg per day, about 0.01mg / kg to about 0.6mg / kg per day, about 0.01mg / kg to about 0.7mg / kg per day, about 0.01mg / kg to about 0.8mg / kg per day, about 0.01mg / kg to about 0.9mg / kg per day ... 0.01mg / kg to about 0.9mg / kg, about 0.01mg / kg to about 1mg / kg per day, about 0.01mg / kg to about 2mg / kg per day, about 0.01mg / kg to about 3mg / kg per day, about 0.01mg / kg to about 4mg / kg per day, about 0.01mg / kg to about 5mg / kg per day, about 0.01mg / kg to about 6mg / kg per day, about 0.01mg / kg to about 7mg / kg per day, about 0.01mg / kg to about 8mg / kg per day, about 0.01mg / kg to about 9mg / kg per day g, about 0.01 mg / kg to about 10 mg / kg per day, about 0.01 mg / kg to about 11 mg / kg per day, about 0.01 mg / kg to about 12 mg / kg per day, about 0.01 mg / kg to about 13 mg / kg per day, about 0.01 mg / kg to about 14 mg / kg per day, about 0.01 mg / kg to about 15 mg / kg per day, about 0.01 mg / kg to about 16 mg / kg per day, about 0.01 mg / kg to about 17 mg / kg per day, about 0.01 mg / kg to about 18 mg / kg per day, about 0.The range is from about 0.01 mg / kg to about 19 mg / kg, or from about 0.01 mg / kg to about 20 mg / kg per day.

[0200] In some embodiments, a therapeutically effective amount of a chimeric protein disclosed herein is from about 0.01 mg / kg to about 0.05 mg / kg per day, from about 0.05 mg / kg to about 0.1 mg / kg per day, from about 0.1 mg / kg to about 0.5 mg / kg per day, from about 0.05 mg / kg to about 1 mg / kg per day, from about 1 mg / kg to about 2 mg / kg per day, from about 2 mg / kg to about 3 mg / kg per day, from about 3 mg / kg to about 4 mg / kg per day, from about 4 mg / kg to about 5 mg / kg per day, from about 5 mg / kg to about 6 mg / kg per day, from about 6 mg / kg to about 7 mg / kg per day, or from about 7 mg / kg to about 8 mg / kg per day. / kg per day, about 8 mg / kg to about 9 mg / kg per day, about 9 mg / kg to about 10 mg / kg per day, about 10 mg / kg to about 11 mg / kg per day, about 11 mg / kg to about 12 mg / kg per day, about 12 mg / kg to about 13 mg / kg per day, about 13 mg / kg to about 14 mg / kg per day, about 14 mg / kg to about 15 mg / kg per day, about 15 mg / kg to about 16 mg / kg per day, about 16 mg / kg to about 17 mg / kg per day, 17 mg / kg to about 18 mg / kg per day, about 18 mg / kg to about 19 mg / kg per day, or about 19 mg / kg to about 20 mg / kg per day.

[0201] In some embodiments, a therapeutically effective amount of a chimeric protein disclosed herein is about 0.01 mg / kg per day, about 0.02 mg / kg per day, about 0.03 mg / kg per day, about 0.04 mg / kg per day, about 0.05 mg / kg per day, about 0.06 mg / kg per day, about 0.07 mg / kg per day, about 0.08 mg / kg per day, about 0.09 mg / kg per day, about 0.1 mg / kg per day, about 0.2 mg / kg per day, about 0.3 mg / kg per day, about 0.4 mg / kg per day, about 0.5 mg / kg per day, about 0.6 mg / kg per day, about 0.7 mg / kg per day, about 0.8 mg / kg per day, about 0. ... about 0.5mg / kg per day, about 0.6mg / kg per day, about 0.7mg / kg per day, about 0.8mg / kg per day, about 0.9mg / kg per day, about 1mg / kg per day, about 1.1mg / kg per day, about 1.2mg / kg per day, about 1.3mg / kg per day, about 1.4mg / kg per day, about 1.5mg / kg per day, about 1.6mg / kg per day, about 1.7mg / kg per day, about 1.8mg / kg per day, about 1.9mg / kg per day, about 2mg / kg per day, about 2.1mg / kg per day About 2.2mg / kg per day, about 2.3mg / kg per day, about 2.4mg / kg per day, about 2.5mg / kg per day, about 2.6mg / kg per day, about 2.7mg / kg per day, about 2.8mg / kg per day, about 2.9mg / kg per day, about 3mg / kg per day, about 3.1mg / kg per day, about 3.2mg / kg per day, about 3.3mg / kg per day, about 3.4mg / kg per day, about 3.5mg / kg per day, about 3.6mg / kg per day, about 3.7mg / kg per day, about 3.8mg / kg, about 3.9mg / kg per day, about 4mg / kg per day, about 4.1mg / kg per day, about 4.2mg / kg per day, about 4.3mg / kg per day, about 4.4mg / kg per day, about 4.5mg / kg per day, about 4.6mg / kg per day, about 4.7mg / kg per day, about 4.8mg / kg per day, about 4.9mg / kg per day, about 5mg / kg per day, about 5.1mg / kg per day, about 5.2mg / kg per day, about 5.3mg / kg per day, about 5.4mg / kg per day, about 5.5mg / kg, about 5.6mg / kg per day, about 5.7mg / kg per day, about 5.8mg / kg per day, about 5.9mg / kg per day, about 6mg / kg per day, about 6.1mg / kg per day, about 6.2mg / kg per day, about 6.3mg / kg per day, about 6.4mg / kg per day, about 6.5mg / kg per day, about 6.6mg / kg per day, about 6.7mg / kg per day, about 6.8mg / kg per day about 6.9mg / kg, about 7mg / kg per day, about 7.1mg / kg per day, about 7.2mg / kg per day, about 7.3mg / kg per day, about 7.4mg / kg per day, about 7.5mg / kg per day, about 7.6mg / kg per day, about 7.7mg / kg per day, about 7.8mg / kg per day, about 7.9mg / kg per day, about 8mg / kg per day, about 8.1mg / kg per day, about 8.2mg / kg per day about 8.3mg / kg, about 8.4mg / kg per day, about 8.5mg / kg per day, about 8.6mg / kg per day, about 8.7mg / kg per day, about 8.8mg / kg per day, about 8.9mg / kg per day, about 9mg / kg per day, about 9.1mg / kg per day, about 9.2mg / kg per day, about 9.3mg / kg per day, about 9.4mg / kg per day, about 9.5mg / kg per day, about 9.6mg / kg per day The range is about 9.7 mg / kg per day, about 9.8 mg / kg per day, about 9.9 mg / kg per day, about 10 mg / kg per day, about 11 mg / kg per day, about 12 mg / kg per day, about 13 mg / kg per day, about 14 mg / kg per day, about 15 mg / kg per day, about 16 mg / kg per day, about 17 mg / kg per day, about 18 mg / kg per day, about 19 mg / kg per day, or about 20 mg / kg per day.

[0202] In some embodiments, an effective dose is administered daily to a subject in need thereof for a period of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days. In some embodiments, an effective dose is administered daily to a subject with an acute CNS disorder for a period of at least 2 to 14 days, e.g., 4, 5, 6, or 7 days. In some embodiments, an effective dose is administered daily to a subject with an acute cardiovascular disorder (e.g., STEMI, cardiac arrest) for a period of at least 2 to 14 days, e.g., 4, 5, 6, or 7 days. In some embodiments, an effective dose is administered daily to a subject with acute kidney injury for a period of at least 2 to 14 days, e.g., 4, 5, 6, or 7 days. In some embodiments, an effective dose is administered daily to a subject experiencing acute radiation sickness (e.g., GI-ARS, H-ARS) for a period of at least 2 to 14 days, e.g., 4, 5, 6, or 7 days. In some embodiments, an effective dose is administered daily for a period of at least 2 days to a period of 14 days, e.g., 4, 5, 6, or 7 days, to a subject experiencing a chemical inhalation injury (e.g., sulfur mustard exposure). In some embodiments, an effective dose is administered daily for a period of at least 1 day to a period of 14 days, e.g., 4, 5, 6, or 7 days, to a subject undergoing a procedure at risk for iatrogenic injury (e.g., transcatheter aortic valve replacement, percutaneous coronary intervention, coronary artery bypass graft surgery). In some embodiments, an effective dose is administered daily for a period of at least 1 day to a period of 14 days, e.g., 4, 5, 6, or 7 days, to a subject undergoing an organ transplant procedure (e.g., skin, kidney, liver, heart, lung). In some embodiments, an effective dose is administered daily for a period of at least 1 day to a period of 14 days, e.g., 4, 5, 6, or 7 days, to a subject undergoing a cosmetic dermatological treatment (e.g., laser resurfacing). In some embodiments, an effective dose is administered to a subject being treated for a dermatological disorder (e.g., wound healing) for a period of at least 1 day to 14 days, e.g., 4, 5, 6, 7 days. In some embodiments, an effective dose is administered to a subject being treated for a traumatic disorder (e.g., burn, crush, laceration, contusion, abrasion, concussion, fracture, amputation) for a period of at least 1 day to 14 days, e.g., 4, 5, 6, 7 days.In some embodiments, the effective dose is administered in an intermittent repeated dose regimen as life-prolonging treatment to a subject experiencing a chronic neurodegenerative disease (e.g., synucleinopathy, amyloidosis).

[0203] In some embodiments, the effective dose is a tapering regimen administered continuously daily to a subject in need thereof for a period of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days. In some embodiments, the tapering regimen reduces the need for supplemental dextrose infusions.

[0204] In some embodiments, an effective dose is administered daily to a subject with a neurovegetative disease for a period of at least 2 days to at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, or 20 years.

[0205] In some embodiments, an effective amount is administered to a subject in need thereof once daily (or every 24 hours). In some embodiments, an effective amount is administered to a subject in need thereof once daily for a period of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days. In some embodiments, an effective amount is administered to a subject in need thereof once daily for a period of up to 2 days, up to 3 days, up to 4 days, up to 5 days, up to 6 days, up to 7 days, up to 8 days, up to 9 days, up to 10 days, up to 11 days, up to 12 days, up to 13 days, up to 14 days. In some embodiments, an effective amount is administered to a subject in need thereof once daily for a period of 7 days. In some embodiments, an effective amount is administered to a subject in need thereof twice, three times, or more times daily. In some embodiments, an effective amount is administered to a subject in need thereof twice, three, or more times per day for a period of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days. In some embodiments, an effective amount is administered to a subject in need thereof twice, three, or more times per day for a period of up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, or up to 14 days. In some embodiments, an effective amount is administered to a subject in need thereof twice, three, or more times per day for a period of 7 days.

[0206] In some embodiments, a total dose of about 5 to 100 mg / kg of a chimeric protein described herein is administered over a 4-day, 5-day, 6-day, or 7-day period. In some embodiments, a total dose of about 5 to 100 mg / kg of a chimeric protein described herein is administered over a 7-day period. In some embodiments, a total dose of about 5 to 90 mg / kg of a chimeric protein described herein is administered over a 4-day, 5-day, 6-day, or 7-day period. In some embodiments, a total dose of about 5 to 90 mg / kg of a chimeric protein described herein is administered over a 7-day period. In some embodiments, a total dose of about 5 to 80 mg / kg of a chimeric protein described herein is administered over a 4-day, 5-day, 6-day, or 7-day period. In some embodiments, a total dose of about 5 to 80 mg / kg of a chimeric protein described herein is administered over a 7-day period. In some embodiments, a total dose of about 5 to 70 mg / kg of a chimeric protein described herein is administered over a 4-day, 5-day, 6-day, or 7-day period. In some embodiments, a total dose of about 5 to 70 mg / kg of a chimeric protein described herein is administered over a 7-day period. In some embodiments, a total dose of about 5 to 60 mg / kg of a chimeric protein described herein is administered over a 4-, 5-, 6-, or 7-day period. In some embodiments, a total dose of about 5 to 60 mg / kg of a chimeric protein described herein is administered over a 7-day period. In some embodiments, a total dose of about 5 to 50 mg / kg of a chimeric protein described herein is administered over a 4-, 5-, 6-, or 7-day period. In some embodiments, a total dose of about 5 to 50 mg / kg of a chimeric protein described herein is administered over a 7-day period. In some embodiments, a total dose of about 5 to 40 mg / kg of a chimeric protein described herein is administered over a 4-, 5-, 6-, or 7-day period. In some embodiments, a total dose of about 5 to 40 mg / kg of a chimeric protein described herein is administered over a 7-day period.In some embodiments, a total dose of about 5 to 30 mg / kg of a chimeric protein described herein is administered over a period of 4, 5, 6, or 7 days. In some embodiments, a total dose of about 5 to 30 mg / kg of a chimeric protein described herein is administered over a period of 7 days. In some embodiments, a total dose of about 5 to 20 mg / kg of a chimeric protein described herein is administered over a period of 4, 5, 6, or 7 days. In some embodiments, a total dose of about 5 to 20 mg / kg of a chimeric protein described herein is administered over a period of 7 days. In some embodiments, a total dose of about 5 to 20 mg / kg of a chimeric protein described herein is administered over a period of 4 days.

[0207] In some embodiments, a total dose of about 100 to 500 mg / kg of a chimeric protein described herein is administered over a 4-, 5-, 6-, or 7-day period. In some embodiments, a total dose of about 100 to 500 mg / kg of a chimeric protein described herein is administered over a 7-day period. In some embodiments, a total dose of about 100 to 400 mg / kg of a chimeric protein described herein is administered over a 4-, 5-, 6-, or 7-day period. In some embodiments, a total dose of about 100 to 400 mg / kg of a chimeric protein described herein is administered over a 7-day period. In some embodiments, a total dose of about 100 to 300 mg / kg of a chimeric protein described herein is administered over a 4-, 5-, 6-, or 7-day period. In some embodiments, a total dose of about 100 to 300 mg / kg of a chimeric protein described herein is administered over a 7-day period. In some embodiments, a total dose of about 100 to 200 mg / kg of a chimeric protein described herein is administered over a 4-, 5-, 6-, or 7-day period. In some embodiments, a total dose of about 100 to 200 mg / kg of a chimeric protein described herein is administered over a 7-day period. In some embodiments, a total dose of about 100 to 150 mg / kg of a chimeric protein described herein is administered over a 4-, 5-, 6-, or 7-day period. In some embodiments, a total dose of about 100 to 150 mg / kg of a chimeric protein described herein is administered over a 7-day period. In some embodiments, a total dose of about 140 mg / kg of a chimeric protein described herein is administered over a 4-, 5-, 6-, or 7-day period. In some embodiments, a total dose of about 140 mg / kg of a chimeric protein described herein is administered over a 7-day period.

[0208] In some embodiments, the treatment regimen comprises a tapering dosing regimen, in which the dose of a chimeric protein of the present disclosure administered to a subject decreases as the treatment period progresses. In some embodiments, the dose administered on day 2 is lower than the dose administered on day 2. In other embodiments, the dose administered on day 2 is the same as the dose administered on day 2, and the dose administered on day 3 is lower than the dose administered on day 2. In some embodiments, the treatment regimen provides a total dose of about 2 mg / kg to about 200 mg / kg, about 2 mg / kg to about 20 mg / kg, about 2 mg / kg to about 10 mg / kg, about 100 mg / kg to about 200 mg / kg, or about 100 mg / kg to about 150 mg / kg over a predetermined period of time (e.g., 4, 5, 6, or 7 days). In some embodiments, the treatment regimen provides a total dose of about 5 mg / kg, about 10 mg / kg, or about 20 mg / kg over a predetermined period of time (eg, 4, 5, 6, or 7 days).

[0209] In some embodiments, a first daily dose is administered on day 1, a second daily dose containing about 85% to about 95% of the amount of chimeric protein present in the first dose is administered on day 2, a third daily dose containing about 65% to about 85% of the amount of chimeric protein present in the second dose but less than the second dose is administered on day 3, and a fourth daily dose containing about 45% to about 65% of the amount of chimeric protein present in the first dose but less than the third dose is administered on day 4. a fifth dose is administered on the fifth day, which is about 35% to about 45% of the amount of chimeric protein present in the first dose but less than the fourth dose; a sixth dose is administered on the sixth day, which is about 25% to about 35% of the amount of chimeric protein present in the first dose but less than the fifth dose; and a seventh dose is administered on the seventh day, which is about 15% to about 25% of the amount of chimeric protein present in the first dose but less than the sixth dose. In some embodiments, the tapering treatment regimen comprises administering a first daily dose on day 1, a second daily dose equivalent to about 90% of the first daily dose on day 2, a third daily dose equivalent to about 70% of the first daily dose on day 3, a fourth daily dose equivalent to about 50% of the first daily dose on day 4, a fifth daily dose equivalent to about 40% of the first daily dose on day 5, a sixth daily dose equivalent to about 30% of the first daily dose on day 6, and a seventh daily dose equivalent to about 20% of the first daily dose on day 7.

[0210] In some embodiments, a first daily dose is administered on day 1, a second daily dose containing about 85% to about 95% (e.g., about 90%) of the amount of chimeric protein present in the first dose is administered on day 2, a third daily dose containing about 65% to about 85% (e.g., about 70%) of the amount of chimeric protein present in the second dose but less than the second dose is administered on day 3, and a fourth dose containing about 45% to about 65% (e.g., about 50%) of the amount of chimeric protein present in the first dose but less than the third dose is administered on day 4.

[0211] In some embodiments, the first daily dose administered on day 1 and the second daily dose administered on day 2 are the same, a third daily dose containing about 65% to about 90% (e.g., about 75%) of the amount of chimeric protein present in the first dose is administered on day 3, and a fourth daily dose containing about 45% to about 65% (e.g., about 50%) of the amount of chimeric protein present in the second dose, but less than the second dose, is administered on day 4.

[0212] In some embodiments, the treatment regimen or tapering treatment regimen provides a total dose of about 2 mg / kg to about 20 mg / kg over a 7-day period. In some embodiments, the treatment regimen provides a total dose of about 2 mg / kg to about 20 mg / kg over a 4-day period. In some embodiments, the treatment regimen or tapering treatment regimen provides a total dose of about 2 mg / kg to about 10 mg / kg over a 7-day period. In some embodiments, the treatment regimen or tapering treatment regimen provides a total dose of about 2 mg / kg to about 10 mg / kg over a 4-day period. In some embodiments, the treatment regimen or tapering treatment regimen provides a total dose of about 5 mg / kg, about 10 mg / kg, or about 20 mg / kg over a 7-day period. In some embodiments, the treatment regimen or tapering treatment regimen provides a total dose of about 5 mg / kg, about 10 mg / kg, or about 20 mg / kg over a 4-day period.

[0213] In some embodiments, the treatment regimen comprises a 5-day course of intravenous administration of a chimeric protein, such as scp776. In some embodiments, the treatment regimen comprises a 7-day course of intravenous administration of a chimeric protein, such as scp776. In some embodiments, the treatment regimen comprises a 4-day course of intravenous administration of a chimeric protein, such as scp776. In some embodiments, the treatment regimen comprises a 3-day course of intravenous administration of a chimeric protein, such as scp776. In some embodiments, the treatment regimen comprises a 2-day course of intravenous administration of a chimeric protein, such as scp776. In some embodiments, the treatment regimen comprises intravenously administering a first dose of about 2 mg / kg to about 6 mg / kg on day 1, and a single dose of about 1 mg / kg to about 2 mg / kg each day thereafter. In some embodiments, the first and second doses are the same (e.g., 2 mg / kg), and the third dose is less than the first dose. For example, in a 4-day treatment course, the first dose can be 2 mg / kg, the second dose can be 2 mg / kg, the third dose can be 1.5 mg / kg, and the fourth dose can be 1 mg / kg. In some embodiments, the second dose is less than the first dose, the third dose can be less than the second dose, etc. For example, in a 4-day treatment course, the first dose can be 2 mg / kg, the second dose can be 1.8 mg / kg, the third dose can be 1.4 mg / kg, and the fourth dose can be 1 mg / kg. In some embodiments, the second dose is less than the first dose, the third dose is the same as the second dose, etc. For example, if the course is a 5-day course, the course can include a first dose of about 5.2 mg / kg on day 1, and a single dose of about 1.3 mg / kg on days 2, 3, 4, and 5.

[0214] In some embodiments, a course of treatment comprises administration of an effective amount for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days. In some embodiments, a course of treatment comprises administration of an effective amount for 7 days. In some embodiments, a course of treatment comprises administration of an effective amount for 4 days. In some embodiments, a course of treatment comprises administration of an effective amount on consecutive days. In some embodiments, a course of treatment comprises administration of an effective amount every day, every two days, every three days, or every four days.

[0215] In some embodiments, human dosing regimens can be calculated based on allometric scaling from non-human primates to human dosing regimens.

[0216] An estimate of the human equivalent dose (HED) of an effective dosing regimen is obtained via allometric scaling (USDHHS, FDA, CDER, Guidance for Industry, 2005). Allometric scaling addresses the issue of differing metabolic rates between species by utilizing a correction factor based on body surface area for the dose of interest in a given species. While allometric scaling is most often used to estimate a safe starting dose for initial human studies, it is also commonly used to convert effective doses from animals to humans. The FDA-recommended correction factor for converting doses from a 3 kg rhesus monkey to humans is 3.1 (USDHHS, FDA, CDER, Guidance for Industry, 2005). In some embodiments, the estimated HED of an effective dosing regimen includes a starting dose of 5.2 mg / kg, followed by booster doses of 1.3 mg / kg spaced 24 hours apart.

[0217] In some embodiments, the pharmaceutical compositions described herein may further comprise one or more additional bioactive or therapeutic substances or components that assist in the treatment of damaged tissue or cells and / or that promote the tissue regeneration process.

[0218] In some embodiments, the method involves administering two or more (e.g., two, three, or more) pharmaceutical compositions. The different pharmaceutical compositions may be administered to the subject in any order and at any suitable interval. For example, in some embodiments, one or more compositions of some embodiments are administered simultaneously or near simultaneously. In some embodiments, the method involves staggered administration of two or more compositions of some embodiments, where a first composition of some embodiments is administered and a second composition of some embodiment is administered at some time thereafter. Any suitable administration interval that produces the desired therapeutic effect may be used.

[0219] In certain embodiments, the method has an additive effect, where the overall effect of administering the combination of therapeutic agents or treatments is approximately equal to the sum of the effects of each therapeutic agent or treatment administered alone, while in other embodiments, the method has a synergistic effect, where the overall effect of administering the combination of therapeutic agents or treatments is greater than the sum of the effects of each therapeutic agent or treatment administered alone.

[0220] In some embodiments, a therapeutically effective amount of a pharmaceutical composition comprising a chimeric protein reduces at least one symptom associated with a disorder, e.g., by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%. In some embodiments, a therapeutically effective amount of a chimeric protein reduces at least one symptom associated with a nervous system disorder, e.g., by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%. In some embodiments, a therapeutically effective amount of a chimeric protein disclosed herein reduces at least one symptom associated with a nervous system disorder by, e.g., about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%. In some embodiments, a therapeutically effective amount of the chimeric protein reduces at least one symptom associated with the disorder, e.g., for at least 1 week, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months.

[0221] Dextrose supplementation Administration of IGF-1 or IGF-1 chimeric proteins (also referred to herein as chimeric proteins, fusion proteins, or IGF-1-containing fusion proteins) has off-target effects that lower blood glucose levels. Aspects of the present disclosure relate to the administration of variable-rate intravenous infusions of dextrose solutions in saline (i.e., 0.9%), semi-normal saline (i.e., 0.45%), lactated Ringer's solution or Hartmann's solution (i.e., 130-131 mM NaCl, 4-5 mM KCl, 2-3 mM CaCl, 28-29 mM C3H5NaO3), 0.2% saline, or water to buffer the blood glucose-lowering effect. In some embodiments, maintenance of normoglycemia is achieved by continuous intravenous dextrose infusion following administration of IGF-1 or an IGF-1-containing fusion protein. In some embodiments, with a slow dextrose infusion, normal blood glucose levels can be maintained following therapeutic administration of IGF-1 or an IGF-1-containing fusion protein. In some embodiments, in the event that blood glucose levels decrease following therapeutic administration of an IGF-1-containing fusion protein: Transient hypoglycemia can be reversed by increasing the rate of supplemental dextrose infusion. In subjects receiving multiple daily doses of IGF-1 or an IGF-1-containing fusion protein, the rate of supplemental dextrose infusion can be gradually reduced and discontinued 24 to 48 hours after the last therapeutic dose of IGF-1 or an IGF-1-containing fusion protein. The euglycemic buffering capacity of supplemental dextrose following administration of an IGF-1-containing fusion protein is not dependent on the exact amino acid sequence of the IGF-1 variant contained within the IGF-1-containing fusion protein or the composition of other domains in the fusion protein (because the blood glucose-lowering capacity is due to the IGF-1 domain).

[0222] Provided herein is a method of dextrose supplementation to support normoglycemia and / or prevent long-term hypoglycemic excursions.

[0223] In some embodiments, the method comprises administering a dextrose solution to a subject in need thereof before, during, and / or after administering IGF-1 or an IGF-1 chimeric protein (or a pharmaceutical composition comprising IGF-1 or an IGF-1 chimeric protein) or any combination thereof to maintain normoglycemia. In some embodiments, the administration of the dextrose solution mitigates the deleterious hypoglycemic effects of administration of IGF-1 or an IGF-1 chimeric protein. In some embodiments, the administration of the dextrose solution is by intravenous infusion. In other embodiments, the administration of the dextrose solution is by intravenous bolus administration.

[0224] In some embodiments, the dextrose solution is administered about 30 minutes, about 25 minutes, about 20 minutes, about 15 minutes, about 10 minutes, or about 5 minutes before administration of the IGF-1 or IGF-1 chimeric protein or pharmaceutical composition comprising the IGF-1 or IGF-1 chimeric protein. In some embodiments, the dextrose solution is administered about 25 to about 30 minutes, about 20 to about 25 minutes, about 15 to about 20 minutes, about 10 to about 15 minutes, or about 5 to about 10 minutes before administration of the IGF-1 or IGF-1 chimeric protein or pharmaceutical composition comprising the IGF-1 or IGF-1 chimeric protein. In some embodiments, a subject in need thereof treated with an IGF-1 or IGF-1 chimeric protein or pharmaceutical composition described herein is continuously infused with a dextrose solution to support normoglycemia. In some embodiments, a subject in need thereof is continuously infused with a dextrose solution for a period of about 24 hours, about 48 hours, about 72 hours, about 96 hours, or about 120 hours. In some embodiments, a subject in need thereof is continuously infused with a dextrose solution for a period of about 24 hours to about 120 hours, about 24 hours to about 96 hours, about 24 hours to about 72 hours, about 24 hours to about 48 hours, about 48 hours to about 120 hours, about 48 hours to 96 hours, about 48 hours to 72 hours, about 72 hours to 120 hours, about 72 hours to about 96 hours, or about 96 hours to about 120 hours. In some embodiments, a subject in need thereof is infused with a dextrose solution about 30 minutes, about 25 minutes, about 20 minutes, about 15 minutes, about 10 minutes, or about 5 minutes before administration of the IGF-1 or IGF-1 chimeric protein, and the dextrose is continuously infused over a period of about 24 hours, about 48 hours, about 72 hours, about 24 to 72 hours, about 24 to 48 hours, or about 48 to 72 hours. In some embodiments, the infusion is for a period of about 48 hours.

[0225] In some embodiments, the subject is infused with dextrose solution for about 48 hours. In some embodiments, the subject is infused with dextrose solution until the end of treatment with IGF-1 or an IGF-1 chimeric protein. In some embodiments, the subject is infused with dextrose solution until about 24 hours after the end of treatment with IGF-1 or an IGF-1 chimeric protein.

[0226] For example, a subject may be treated with a pharmaceutical composition comprising IGF-1 or an IGF-1 chimeric protein for four days, and the subject may be infused with a dextrose solution for up to four or five days. Alternatively, a subject may be treated with a pharmaceutical composition comprising IGF-1 or an IGF-1 chimeric protein for one, two, or three days, and the subject may be infused with a dextrose solution for up to two, three, or four days. Alternatively, a subject may be treated daily with a pharmaceutical composition comprising IGF-1 or an IGF-1 chimeric protein for up to five days, and the subject may be infused with a dextrose solution for up to one or two days following the last treatment with a pharmaceutical composition comprising IGF-1 or an IGF-1 chimeric protein.

[0227] In some embodiments, a subject in need thereof is administered a bolus dose of IGF-1 or an IGF-1 chimeric protein or a pharmaceutical composition comprising an IGF-1 or an IGF-1 chimeric protein, and a dextrose solution is administered by continuous intravenous infusion. In some embodiments, a subject in need thereof is administered an intrathecal dose of IGF-1 or an IGF-1 chimeric protein or a pharmaceutical composition comprising an IGF-1 or an IGF-1 chimeric protein, and a dextrose solution is administered by continuous intravenous infusion. In some embodiments, the administration of the dextrose solution by continuous infusion occurs about 1 to about 30 minutes before the administration of the IGF-1 or IGF-1 chimeric protein. In some embodiments, the administration of the dextrose solution by continuous infusion occurs about 30 minutes, about 25 minutes, about 20 minutes, about 15 minutes, about 10 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, or about 1 minute before the administration of the IGF-1 or IGF-1 chimeric protein. In some embodiments, the administration of the dextrose solution by continuous infusion is carried out during the administration of IGF-1 or an IGF-1 chimeric protein or a pharmaceutical composition comprising an IGF-1 or an IGF-1 chimeric protein. In some embodiments, the administration of the dextrose solution by continuous infusion is carried out during and after the administration of IGF-1 or an IGF-1 chimeric protein or a pharmaceutical composition comprising an IGF-1 or an IGF-1 chimeric protein. In some embodiments, the pharmaceutical composition comprising an IGF-1 or an IGF-1 chimeric protein does not contain dextrose.

[0228] In some embodiments, the method includes initiating a dextrose infusion, beginning shortly (t≦30 minutes) after administration of the IGF-1 or IGF-1-containing fusion protein at a slow rate (i.e., 0.1 mL / kg / h). In some embodiments, the method further includes adjusting the infusion rate to support euglycemia.

[0229] In some embodiments, the infusion may be at a rate ranging from 0.01 mL / kg / hr to 5 mL / kg / hr. For example, the infusion may be at a rate of about 0.1 mL / kg / hr. In some embodiments, the infusion may begin at a rate of about 0.1 mL / kg / hr, followed by an increment of +0.05 mL / kg / hr to +2 mL / kg / hr (e.g., +0.05 mL / kg / hr, +0.1 mL / kg / hr, +0.2 mL / kg / hr, +0.3 mL / kg / hr, +0.4 mL / kg / hr, +0.5 mL / kg / hr, +0.6 mL / kg / hr, +0.7 mL / kg / hr, or kg / hr, +0.8mL / kg / hr, +0.9mL / kg / hr, +1mL / kg / hr, +1.1mL / kg / hr, +1.2mL / kg / hr, +1.3mL / kg / hr, +1.34mL / kg / hr, +1.5mL / kg / hr, +1.6mL / kg / hr, +1.7mL / kg / hr, +1.8mL / kg / hr, +1.9mL / kg / hr, +2mL / kg / hr). In some embodiments, the infusion may be at a rate of about 2 mL / kg / hr, followed by decreasing rates of -0.05 mL / kg / hr to -1 mL / kg / hr (e.g., -0.05 mL / kg / hr, -0.1 mL / kg / hr, -0.2 mL / kg / hr, -0.3 mL / kg / hr, -0.4 mL / kg / hr, -0.5 mL / kg / hr, -0.6 mL / kg / hr, -0.7 mL / kg / hr, -0.8 mL / kg / hr, -0.9 mL / kg / hr, -1 mL / kg / hr).

[0230] In some embodiments, the dextrose solution is in sterile water or saline. In some embodiments, the dextrose solution comprises about 5% (w / v) dextrose to about 50% (w / v) dextrose to support normoglycemia. In some embodiments, the dextrose solution is in sterile water or saline. In some embodiments, a subject in need thereof may be administered 5% to 40% dextrose (w / v). In some embodiments, a subject in need thereof may be administered 5% to 35% dextrose (w / v). In some embodiments, a subject in need thereof may be administered 5% to 30% dextrose (w / v). In some embodiments, a subject in need thereof may be administered 5% to 25% dextrose (w / v). In some embodiments, a subject in need thereof may be administered 5% to 20% dextrose (w / v). In some embodiments, a subject in need thereof may be administered 5% to 15% dextrose (w / v), hi some embodiments, a subject in need thereof may be administered 5% to 10% dextrose (w / v).

[0231] In some embodiments, the dextrose solution comprises about 5% (w / v) dextrose, about 6% (w / v) dextrose, about 7% (w / v) dextrose, about 8% (w / v) dextrose, about 9% (w / v) dextrose, about 10% (w / v) dextrose, about 11% (w / v) dextrose, about 12% (w / v) dextrose, about 13% (w / v) dextrose, about 14% (w / v) dextrose, about 15% (w / v) dextrose, about 16% (w / v) dextrose, about 17% (w / v) dextrose, about 19% (w / v) dextrose, or about 20% (w / v) dextrose.

[0232] In some embodiments, the dextrose solution is from about 5% (w / v) dextrose to about 6% (w / v) dextrose, from about 6% (w / v) dextrose to about 7% (w / v) dextrose, from about 7% (w / v) dextrose to about 8% (w / v) dextrose, from about 8% (w / v) dextrose to about 9% (w / v) dextrose, from about 9% (w / v) dextrose to about 10% (w / v) dextrose, from about 10% (w / v) dextrose to about 11% (w / v) dextrose, from about 11% (w / v) dextrose to about 12% (w / v) dextrose, or from about 12% (w / v) dextrose. The range of dextrose concentrations includes about 13% (w / v) dextrose to about 14% (w / v) dextrose, about 14% (w / v) dextrose to about 15% (w / v) dextrose, about 15% (w / v) dextrose to about 16% (w / v) dextrose, about 16% (w / v) dextrose to about 17% (w / v) dextrose, about 17% (w / v) dextrose to about 18% (w / v) dextrose, about 18% (w / v) dextrose to about 19% (w / v) dextrose, or about 19% (w / v) dextrose to about 20% (w / v) dextrose.

[0233] In some embodiments, a subject in need thereof is continuously supplemented with about 5% dextrose to about 20% dextrose (w / v), about 5% dextrose to about 15% dextrose (w / v), about 5% dextrose to about 10% dextrose (w / v), about 5% (w / v), about 10% dextrose (w / v).

[0234] In some embodiments, for rapid adjustment, a 50% (w / v) solution of dextrose may be administered as an IV bolus.

[0235] In some embodiments, blood glucose is monitored during administration of the dextrose solution. In some embodiments, blood glucose is monitored before administration of the dextrose solution. In some embodiments, blood glucose is monitored after administration of the dextrose solution. In some embodiments, blood glucose is monitored every 60 minutes + / - 20 minutes during administration of the dextrose solution. In some embodiments, blood glucose is monitored every 60 minutes + / - 20 minutes during the first 24 hours of dextrose supplementation. In some embodiments, blood glucose is monitored every 120 minutes + / - 20 minutes 24 to 48 hours or more after initiation of dextrose supplementation. In some embodiments, blood glucose is monitored according to standard care beginning 48 hours after initiation of dextrose supplementation. In some embodiments, the rate of dextrose supplementation is reduced or discontinued if the subject's blood glucose measures ≧150 mg / dL. In other embodiments, the rate of dextrose supplementation is reduced or stopped when the subject's blood glucose is measured to be ≧180 mg / dL.

[0236] In some embodiments, the blood glucose management plan (BGMP) follows the current American Heart Association (AHA) / American Stroke Association (ASA) guidance for blood glucose management in acute ischemic stroke (Powers et al, Guidelines for the Early Management of Patients With Acute Ischemic Stroke: 2019 Update to the 2018 Guidelines for the Early Management of Acute Ischemic Stroke: A Guideline for Healthcare Professionals From the American Heart Association / American Stroke Association. Stroke. 2019).

[0237] In some embodiments, a subject in need thereof has their blood glucose levels monitored. In some embodiments, blood glucose (BG) monitoring is performed for a minimum of 48 hours with the following guidelines:

[0238] BG levels are used to determine the rate of dextrose supplementation based on the table below. [Table 1] Abbreviations: BG = blood glucose; BGMP = blood glucose management plan; D10 = 10% dextrose in water

[0239] After each administration of IGF-1 or IGF-1 chimeric protein, the subject's blood glucose level is tested 30 minutes (±15 minutes), and then hourly (±20 minutes) for up to 8 hours after administration. After 8 hours, if blood glucose levels are stable, glucose testing may be changed to every 2 hours (±20 minutes). A stable blood glucose level is defined as a blood glucose level above 100 mg / dL for at least 4 consecutive hours without the need to increase the rate of dextrose infusion.

[0240] Any change in clinical status suggesting a decrease in blood glucose levels (e.g., change in mental status after administration) requires immediate glucose testing and, if no longer stable, a return to hourly testing and adjustment of dextrose rate until stable. If blood glucose levels were stable but are no longer stable: If BG < 80 mg / dL, recheck BG every 15 minutes and adjust / administer dextrose according to Table 5 until BG is confirmed to be > 80 mg / dL. When BG > 80 mg / dose is confirmed, return to hourly glucose testing within 8 hours of administration. If more than 8 hours post-dose, return to hourly glucose testing until BG is stable (BG > 100 mg / dL for 4 consecutive hours without increasing dextrose administration), then glucose testing may be changed again to every 2 hours.

[0241] In some embodiments, BG levels can be measured by central laboratory testing, bedside testing (e.g., i-STAT (登録商標) blood gases), or measured by fingerstick.

[0242] In some embodiments, an IV infusion of dextrose solution (eg, D10) is initiated at a rate of 0.1 mL / kg / h prior to administration of IGF-1 or an IGF-1 chimeric protein.

[0243] In some embodiments, administration of the dextrose solution begins 30 minutes or less prior to injection of the IGF-1 or IGF-1 chimeric protein.

[0244] In some embodiments, administration of the dextrose solution continues for at least 48 hours if the BG level does not exceed 150 mg / dL on two consecutive readings at least 30 minutes apart, in which case dextrose administration is discontinued or the rate of dextrose administration is reduced.

[0245] In some embodiments, if BG levels are falling or are below 80 mg / dL, the rate of administration of the dextrose solution is increased.

[0246] In some embodiments, the dextrose solution may be 10% dextrose in water. In some embodiments, if there is clinical concern about administering excess free water, the dextrose solution may be switched to 5% dextrose in saline at the discretion of the site's clinical trial team.

[0247] In some embodiments, if a 50% dextrose infusion is given for any reason, BG tests (fingerstick or lab glucose) should be performed every 15 minutes until the BG level is >80 mg / dL.

[0248] Additional Embodiments The following additional embodiments are provided for illustrative purposes.

[0249] Embodiment 1: A method of treating a subject in need thereof with an IGF-1 chimeric protein, the method comprising: administering a solution comprising an effective amount of dextrose prior to administering the IGF-1 chimeric protein, wherein the administration is over a period of about 48 hours; and administering a pharmaceutical composition comprising an effective amount of an IGF-1 chimeric protein; Including, The method wherein an effective amount of dextrose supports normoglycemia.

[0250] Embodiment 2: The method of embodiment 1, wherein a solution comprising an effective amount of dextrose is administered by infusion to a subject in need thereof.

[0251] Embodiment 3: The method of embodiment 2, comprising administering a solution comprising an effective amount of dextrose at a starting infusion rate of about 0.1 mL / kg / h.

[0252] Embodiment 4: The method of embodiment 3, further comprising adjusting the infusion rate in increments of +0.05 mL / kg / hr to +2 mL / kg / hr.

[0253] Embodiment 5: The method of any one of the preceding embodiments, wherein the solution comprising an effective amount of dextrose comprises about 5% to about 10% dextrose (w / v) in water or a saline solution.

[0254] Embodiment 6: The method of any one of the preceding embodiments, wherein the solution comprising an effective amount of dextrose is administered from about 1 minute to about 30 minutes prior to administration of the IGF-1 chimeric protein.

[0255] Embodiment 7: The method of any one of the preceding embodiments, wherein the IGF-1 chimeric protein comprises a targeting domain comprising human annexin 5 (AnxV) or a variant thereof, and an activator domain comprising insulin-like growth factor 1 (IGF-1) or a variant thereof.

[0256] Embodiment 8: The method of any one of the preceding embodiments, wherein the IGF-1 chimeric protein further comprises a peptide, wherein the peptide extends the half-life of the IGF-1 chimeric protein.

[0257] Embodiment 9: The method of embodiment 7 or 8, wherein the targeting domain is a non-internalizing variant of annexin 5.

[0258] Embodiment 10: The method of any one of the preceding embodiments, wherein the IGF-1 chimeric protein is not substantially internalized by the cell.

[0259] Embodiment 11: The method of any one of the preceding embodiments, wherein the IGF-1 chimeric protein comprises a non-internalizing variant of annexin 5, wherein the non-internalizing variant of annexin 5 comprises one or more mutations, wherein the one or more mutations comprise a substitution at a position corresponding to C316 and, optionally, one or more positions corresponding to R63, K70, K101, E138, D139, N160, and combinations thereof.

[0260] Embodiment 12: The method of any one of embodiments 7-11, wherein the activator domain of the IGF-1 chimeric protein is a variant of human insulin-like growth factor IGF-1 comprising one or more mutations, wherein the one or more mutations consist of substitutions at one or more positions corresponding to E3, Y24, Y31, Y60, and combinations thereof.

[0261] Embodiment 13: The method of any one of the preceding embodiments, wherein the IGF-1 chimeric protein further comprises a half-life modulator comprising a variant of human serum albumin (HSA) comprising one or more mutations, wherein the one or more mutations consist of substitutions at one or more positions corresponding to C58 and N527, and combinations thereof.

[0262] Embodiment 14: The method of any one of the preceding embodiments, wherein the IGF-1 chimeric protein comprises or consists of IGF1(E3R / Y31A)_lk7_HSA26-609(C58S / N527Q)_lk7_AnxV2-320(R63A / K70A / K101A / E138A / D139G / N160A / C316A).

[0263] Embodiment 15: The method of any one of the preceding embodiments, wherein the IGF-1 chimeric protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24.

[0264] Embodiment 16: The method of any one of the preceding embodiments, comprising administering an effective amount of an IGF-1 chimeric protein in a tapered manner over a period of 2 to 14 days or longer.

[0265] Embodiment 17: The method of any one of the preceding embodiments, wherein administering an effective amount of an IGF-1 chimeric protein inhibits apoptosis.

[0266] Embodiment 18: The method of any one of the preceding embodiments, wherein the pharmaceutical composition further comprises at least one physiologically acceptable carrier.

[0267] Embodiment 19: The method of any one of the preceding embodiments, wherein the subject in need thereof is a human.

[0268] Embodiment 20: The method of any one of the preceding embodiments, wherein the subject in need thereof has an acute CNS disorder or an acute cardiovascular disorder (e.g., STEMI, cardiac arrest).

[0269] Embodiment 21: The method of any one of the preceding embodiments, wherein the subject in need thereof has acute radiation sickness (e.g., GI-ARS, H-ARS).

[0270] Embodiment 22: The method of any one of the preceding embodiments, wherein the subject in need thereof has a chemical inhalation injury (e.g., sulfur mustard exposure).

[0271] Embodiment 23: The method of any one of the preceding embodiments, wherein the subject in need thereof has a dermatological disorder (e.g., wound healing).

[0272] Embodiment 24: The method of any one of the preceding embodiments, wherein the subject in need thereof has a traumatic injury (e.g., a burn, crush, laceration, contusion, avulsion, concussion, fracture, amputation).

[0273] Embodiment 25: The method of any one of the preceding embodiments, wherein the subject in need thereof has a chronic neurodegenerative disease (e.g., synucleinopathy, amyloidosis).

[0274] Embodiment 26: The method of any one of the preceding embodiments, wherein the subject in need thereof is undergoing a procedure (e.g., transcatheter aortic valve replacement, percutaneous coronary intervention, coronary artery bypass surgery) that carries a risk of iatrogenic injury.

[0275] Embodiment 27: The method of any one of the preceding embodiments, wherein the subject in need thereof is undergoing an organ transplant procedure (e.g., skin, kidney, liver, heart, lung).

[0276] Embodiment 28: The method of any one of the preceding embodiments, wherein the subject in need thereof is undergoing a cosmetic dermatological treatment (e.g., laser resurfacing).

[0277] Embodiment 29: An IGF-1 chimeric protein for use in a method of treatment of a subject in need thereof, the method comprising: administering a solution comprising an effective amount of dextrose prior to administering the IGF-1 chimeric protein, wherein the administration is for a period of about 48 hours; and administering a pharmaceutical composition comprising an effective amount of the IGF-1 chimeric protein. Including, An IGF-1 chimeric protein in which an effective amount of dextrose supports normoglycemia.

[0278] Embodiment 30: Dextrose for use in a method of treatment of a subject in need thereof with an IGF-1 chimeric protein, the method comprising: administering a solution comprising an effective amount of said dextrose prior to administering an IGF-1 chimeric protein, wherein the administration is for a period of about 48 hours; and administering a pharmaceutical composition comprising an effective amount of an IGF-1 chimeric protein. Including, Dextrose, an effective amount of dextrose supports normoglycemia.

[0279] Embodiment 31: A combination of an IGF-1 chimeric protein and dextrose for use in a method of treatment of a subject in need thereof, the method comprising: administering a solution comprising an effective amount of dextrose prior to administering the IGF-1 chimeric protein, wherein the administration is for a period of about 48 hours; and administering a pharmaceutical composition comprising an effective amount of the IGF-1 chimeric protein. Including, A combination in which an effective amount of dextrose supports normoglycemia.

[0280] Embodiment 32: The IGF-1 chimeric protein, dextrose, or a combination of IGF-1 chimeric protein and dextrose according to embodiments 29-31, wherein a solution containing an effective amount of dextrose is administered by infusion to a subject in need thereof.

[0281] Embodiment 33: The IGF-1 chimeric protein, dextrose, or a combination of IGF-1 chimeric protein and dextrose of embodiment 32, comprising administering a solution comprising an effective amount of dextrose at a starting infusion rate of about 0.1 mL / kg / h.

[0282] Embodiment 34: The IGF-1 chimeric protein, dextrose, or combination of IGF-1 chimeric protein and dextrose of embodiment 33, further comprising adjusting the infusion rate by an increment of +0.05 mL / kg / hr to +2 mL / kg / hr.

[0283] Embodiment 35: An IGF-1 chimeric protein, dextrose, or a combination of an IGF-1 chimeric protein and dextrose according to embodiments 29-34, wherein the solution containing an effective amount of dextrose comprises about 5% to about 10% dextrose (w / v) in water or saline solution.

[0284] Embodiment 36: An IGF-1 chimeric protein, dextrose, or a combination of an IGF-1 chimeric protein and dextrose according to embodiments 29-35, wherein a solution containing an effective amount of dextrose is administered from about 1 minute to about 30 minutes before administration of the IGF-1 chimeric protein.

[0285] Embodiment 37: An IGF-1 chimeric protein, dextrose, or a combination of an IGF-1 chimeric protein and dextrose according to embodiments 29-36, wherein the IGF-1 chimeric protein comprises a targeting domain comprising human annexin 5 (AnxV) or a variant thereof, and an activator domain comprising insulin-like growth factor 1 (IGF-1) or a variant thereof.

[0286] Embodiment 38: An IGF-1 chimeric protein, dextrose or a combination of an IGF-1 chimeric protein and dextrose according to embodiments 29-37, wherein the IGF-1 chimeric protein further comprises a peptide, and the peptide extends the half-life of the IGF-1 chimeric protein.

[0287] Embodiment 39: The IGF-1 chimeric protein, dextrose or a combination of IGF-1 chimeric protein and dextrose according to embodiment 37 or 38, wherein the targeting domain is a non-internalizing variant of annexin 5.

[0288] Embodiment 40: An IGF-1 chimeric protein, dextrose or a combination of an IGF-1 chimeric protein and dextrose according to embodiments 29-39, wherein the IGF-1 chimeric protein is not substantially internalized by cells.

[0289] Embodiment 41: An IGF-1 chimeric protein, dextrose or a combination of an IGF-1 chimeric protein and dextrose according to embodiments 29-40, wherein the IGF-1 chimeric protein comprises a non-internalizing variant of annexin 5, and the non-internalizing variant of annexin 5 comprises one or more mutations, and the one or more mutations comprise a substitution at a position corresponding to C316 and, optionally, at one or more positions corresponding to R63, K70, K101, E138, D139, N160, and combinations thereof.

[0290] Embodiment 42: An IGF-1 chimeric protein, dextrose or a combination of an IGF-1 chimeric protein and dextrose according to embodiments 37-41, wherein the activator domain of the IGF-1 chimeric protein is a variant of human insulin-like growth factor IGF-1 comprising one or more mutations, wherein the one or more mutations consist of substitutions at one or more positions corresponding to E3, Y24, Y31, Y60 and combinations thereof.

[0291] Embodiment 43: An IGF-1 chimeric protein, dextrose or a combination of an IGF-1 chimeric protein and dextrose according to embodiments 29-42, wherein the IGF-1 chimeric protein further comprises a half-life modulator comprising a variant of human serum albumin (HSA) comprising one or more mutations, wherein the one or more mutations consist of substitutions at one or more positions corresponding to C58 and N527, and combinations thereof.

[0292] Embodiment 44: An IGF-1 chimeric protein, dextrose or a combination of an IGF-1 chimeric protein and dextrose according to embodiments 29-43, wherein the IGF-1 chimeric protein comprises or consists of IGF1(E3R / Y31A)_lk7_HSA26-609(C58S / N527Q)_lk7_AnxV2-320(R63A / K70A / K101A / E138A / D139G / N160A / C316A).

[0293] Embodiment 45: An IGF-1 chimeric protein, dextrose or a combination of an IGF-1 chimeric protein and dextrose according to embodiments 29-44, wherein the IGF-1 chimeric protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24.

[0294] Embodiment 46: An IGF-1 chimeric protein, dextrose, or a combination of an IGF-1 chimeric protein and dextrose according to embodiments 29-45, comprising administering an effective amount of an IGF-1 chimeric protein in a tapered manner over a period of 2 to 14 days or longer.

[0295] Embodiment 47: The IGF-1 chimeric protein, dextrose, or a combination of IGF-1 chimeric protein and dextrose of embodiments 29-46, wherein administering an effective amount of the IGF-1 chimeric protein inhibits apoptosis.

[0296] Embodiment 48: The IGF-1 chimeric protein, dextrose or a combination of IGF-1 chimeric protein and dextrose according to embodiments 29-47, wherein the pharmaceutical composition further comprises at least one physiologically acceptable carrier.

[0297] Embodiment 49: The IGF-1 chimeric protein, dextrose or a combination of IGF-1 chimeric protein and dextrose according to embodiments 29-48, wherein the subject in need thereof is a human.

[0298] Embodiment 50: The IGF-1 chimeric protein, dextrose, or a combination of IGF-1 chimeric protein and dextrose according to embodiments 29-49, wherein the subject in need thereof has an acute CNS disorder or an acute cardiovascular disorder (e.g., STEMI, cardiac arrest).

[0299] Embodiment 51: The IGF-1 chimeric protein, dextrose, or a combination of IGF-1 chimeric protein and dextrose according to embodiments 29-50, wherein the subject in need thereof has acute radiation sickness (e.g., GI-ARS, H-ARS).

[0300] Embodiment 52: The IGF-1 chimeric protein, dextrose, or a combination of IGF-1 chimeric protein and dextrose according to embodiments 29-51, wherein the subject in need thereof has a chemical inhalation injury (e.g., sulfur mustard exposure).

[0301] Embodiment 53: The IGF-1 chimeric protein, dextrose, or a combination of IGF-1 chimeric protein and dextrose according to embodiments 29-52, wherein the subject in need thereof has a dermatological disorder (e.g., wound healing).

[0302] Embodiment 54: The IGF-1 chimeric protein, dextrose, or a combination of IGF-1 chimeric protein and dextrose described in embodiments 29-53, wherein the subject in need thereof has a traumatic injury (e.g., a burn, crush, laceration, contusion, avulsion, concussion, fracture, amputation).

[0303] Embodiment 55: The IGF-1 chimeric protein, dextrose, or a combination of IGF-1 chimeric protein and dextrose according to embodiments 29-54, wherein the subject in need thereof has a chronic neurodegenerative disease (e.g., synucleinopathy, amyloidosis).

[0304] Embodiment 56: The IGF-1 chimeric protein, dextrose, or combination of IGF-1 chimeric protein and dextrose described in embodiments 29-55, wherein a subject in need thereof is undergoing a procedure that carries a risk of iatrogenic injury (e.g., transcatheter aortic valve replacement, percutaneous coronary intervention, coronary artery bypass surgery).

[0305] Embodiment 57: The IGF-1 chimeric protein, dextrose, or a combination of IGF-1 chimeric protein and dextrose described in embodiments 29-56, wherein the subject in need thereof is undergoing organ transplantation treatment (e.g., skin, kidney, liver, heart, lung).

[0306] Embodiment 58: The IGF-1 chimeric protein, dextrose, or a combination of IGF-1 chimeric protein and dextrose according to embodiments 29-57, wherein the subject in need thereof is undergoing a cosmetic dermatological treatment (e.g., laser resurfacing).

[0307] Embodiment 59: Use of an IGF-1 chimeric protein in the manufacture of a medicament for the treatment of a subject in need thereof, wherein a solution comprising an effective amount of dextrose is administered prior to administering a pharmaceutical composition comprising an effective amount of an IGF-1 chimeric protein, and wherein administration of the solution is over a period of about 48 hours, and wherein the effective amount of dextrose supports normoglycemia.

[0308] Embodiment 60: Use of dextrose and an IGF-1 chimeric protein in the manufacture of a medicament for treating a subject in need thereof, wherein a solution comprising an effective amount of dextrose is administered prior to administering a pharmaceutical composition comprising an effective amount of an IGF-1 chimeric protein, and wherein the administration of the solution comprising the effective amount of dextrose is for a period of about 48 hours, and wherein the effective amount of dextrose supports normoglycemia.

[0309] Embodiment 61: Use of a combination of an IGF-1 chimeric protein and dextrose in the manufacture of a medicament for the treatment of a subject in need thereof, wherein a solution comprising an effective amount of dextrose is administered prior to administering a pharmaceutical composition comprising an effective amount of an IGF-1 chimeric protein, and wherein the administration of the solution comprising the effective amount of dextrose is for a period of about 48 hours, and wherein the effective amount of dextrose supports normoglycemia.

[0310] Embodiment 63: An IGF-1 chimeric protein, dextrose, or a combination of an IGF-1 chimeric protein and dextrose according to embodiments 59-61, wherein a solution containing an effective amount of dextrose is administered by infusion to a subject in need thereof.

[0311] Embodiment 63: An IGF-1 chimeric protein, dextrose, or a combination of an IGF-1 chimeric protein and dextrose according to embodiment 62, comprising administering a solution comprising an effective amount of dextrose at a starting infusion rate of about 0.1 mL / kg / h.

[0312] Embodiment 64: The IGF-1 chimeric protein, dextrose, or combination of IGF-1 chimeric protein and dextrose of embodiment 63, further comprising adjusting the infusion rate in increments of +0.05 mL / kg / hr to +2 mL / kg / hr.

[0313] Embodiment 65: An IGF-1 chimeric protein, dextrose, or a combination of an IGF-1 chimeric protein and dextrose according to embodiments 59-64, wherein the solution containing an effective amount of dextrose comprises about 5% to about 10% dextrose (w / v) in water or saline solution.

[0314] Embodiment 66: An IGF-1 chimeric protein, dextrose, or a combination of an IGF-1 chimeric protein and dextrose according to embodiments 59-65, wherein a solution containing an effective amount of dextrose is administered from about 1 minute to about 30 minutes before administration of the IGF-1 chimeric protein.

[0315] Embodiment 67: An IGF-1 chimeric protein, dextrose, or a combination of an IGF-1 chimeric protein and dextrose according to embodiments 59-66, wherein the IGF-1 chimeric protein comprises a targeting domain comprising human annexin 5 (AnxV) or a variant thereof, and an activator domain comprising insulin-like growth factor 1 (IGF-1) or a variant thereof.

[0316] Embodiment 68: An IGF-1 chimeric protein, dextrose or a combination of an IGF-1 chimeric protein and dextrose according to embodiments 59-67, wherein the IGF-1 chimeric protein further comprises a peptide, and the peptide extends the half-life of the IGF-1 chimeric protein.

[0317] Embodiment 69: The IGF-1 chimeric protein, dextrose or a combination of IGF-1 chimeric protein and dextrose according to embodiment 67 or 68, wherein the targeting domain is a non-internalizing variant of annexin 5.

[0318] Embodiment 70: An IGF-1 chimeric protein, dextrose or a combination of an IGF-1 chimeric protein and dextrose according to embodiments 59-69, wherein the IGF-1 chimeric protein is not substantially internalized by cells.

[0319] Embodiment 71: An IGF-1 chimeric protein, dextrose or a combination of an IGF-1 chimeric protein and dextrose according to embodiments 59-70, wherein the IGF-1 chimeric protein comprises a non-internalizing variant of annexin 5, and the non-internalizing variant of annexin 5 comprises one or more mutations, and the one or more mutations comprise a substitution at a position corresponding to C316 and, optionally, at one or more positions corresponding to R63, K70, K101, E138, D139, N160, and combinations thereof.

[0320] Embodiment 72: An IGF-1 chimeric protein, dextrose or a combination of an IGF-1 chimeric protein and dextrose according to embodiments 59-71, wherein the activator domain of the IGF-1 chimeric protein is a variant of human insulin-like growth factor IGF-1 comprising one or more mutations, wherein the one or more mutations consist of substitutions at one or more positions corresponding to E3, Y24, Y31, Y60 and combinations thereof.

[0321] Embodiment 73: An IGF-1 chimeric protein, dextrose or a combination of an IGF-1 chimeric protein and dextrose according to embodiments 59-71, wherein the IGF-1 chimeric protein further comprises a half-life modulator comprising a variant of human serum albumin (HSA) comprising one or more mutations, wherein the one or more mutations consist of substitutions at one or more positions corresponding to C58 and N527, and combinations thereof.

[0322] Embodiment 74: An IGF-1 chimeric protein, dextrose or a combination of an IGF-1 chimeric protein and dextrose according to embodiments 59-74, wherein the IGF-1 chimeric protein comprises or consists of IGF1(E3R / Y31A)_lk7_HSA26-609(C58S / N527Q)_lk7_AnxV2-320(R63A / K70A / K101A / E138A / D139G / N160A / C316A).

[0323] Embodiment 75: An IGF-1 chimeric protein, dextrose or a combination of an IGF-1 chimeric protein and dextrose according to embodiments 59-74, wherein the IGF-1 chimeric protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24.

[0324] Embodiment 76: An IGF-1 chimeric protein, dextrose, or a combination of an IGF-1 chimeric protein and dextrose according to embodiments 59-75, comprising administering an effective amount of an IGF-1 chimeric protein in a tapered manner over a period of 2 to 14 days or longer.

[0325] Embodiment 77: The IGF-1 chimeric protein, dextrose, or a combination of IGF-1 chimeric protein and dextrose of embodiments 59-76, wherein administering an effective amount of the IGF-1 chimeric protein inhibits apoptosis.

[0326] Embodiment 78: The IGF-1 chimeric protein, dextrose or a combination of IGF-1 chimeric protein and dextrose according to embodiments 59-77, wherein the pharmaceutical composition further comprises at least one physiologically acceptable carrier.

[0327] Embodiment 79: The IGF-1 chimeric protein, dextrose or a combination of IGF-1 chimeric protein and dextrose according to embodiments 59-78, wherein the subject in need thereof is a human.

[0328] Embodiment 80: The IGF-1 chimeric protein, dextrose, or a combination of IGF-1 chimeric protein and dextrose according to embodiments 59-79, wherein the subject in need thereof has an acute CNS disorder or an acute cardiovascular disorder (e.g., STEMI, cardiac arrest).

[0329] Embodiment 81: The IGF-1 chimeric protein, dextrose, or a combination of IGF-1 chimeric protein and dextrose according to embodiments 59-80, wherein the subject in need thereof has acute radiation sickness (e.g., GI-ARS, H-ARS).

[0330] Embodiment 82: The IGF-1 chimeric protein, dextrose, or a combination of IGF-1 chimeric protein and dextrose according to embodiments 59-81, wherein the subject in need thereof has a chemical inhalation injury (e.g., sulfur mustard exposure).

[0331] Embodiment 83: The IGF-1 chimeric protein, dextrose, or a combination of IGF-1 chimeric protein and dextrose according to embodiments 59-82, wherein the subject in need thereof has a dermatological disorder (e.g., wound healing).

[0332] Embodiment 84: The IGF-1 chimeric protein, dextrose, or a combination of IGF-1 chimeric protein and dextrose described in embodiments 59-83, wherein the subject in need thereof has a traumatic injury (e.g., a burn, crush, laceration, contusion, avulsion, concussion, fracture, amputation).

[0333] Embodiment 85: The IGF-1 chimeric protein, dextrose, or a combination of IGF-1 chimeric protein and dextrose according to embodiments 59-84, wherein the subject in need thereof has a chronic neurodegenerative disease (e.g., synucleinopathy, amyloidosis).

[0334] Embodiment 86: The IGF-1 chimeric protein, dextrose, or a combination of IGF-1 chimeric protein and dextrose described in embodiments 59-85, wherein a subject in need thereof is undergoing a procedure that carries a risk of iatrogenic damage (e.g., transcatheter aortic valve replacement, percutaneous coronary intervention, coronary artery bypass surgery). [Example]

[0335] The following examples are offered by way of illustration and not by way of limitation. Unless otherwise specified, all reagents and solvents are of standard commercial grade and are used without further purification. Using routine modification, the procedures provided in the following examples may be adapted by those of skill in the art to make and use other bispecific proteins and pharmaceutical compositions within the scope of this disclosure.

[0336] Example 1: Effect of intravenous scp776 (60 mg / kg) on ​​blood glucose in healthy C57BL / 6 mice The purpose of this study was to characterize blood glucose profiles after intended high-dose administration of intravenous (i.v.) scp776 in C57BL / 6 mice. These profiles will serve as a baseline for the response to high-dose scp776 administration and will be used for comparison when developing blood glucose supplementation strategies. If deemed appropriate, blood glucose supplementation will be performed in efficacy studies.

[0337] Overview:

[0338] scp776 (mg / mL in 20 mM Tris, 7.5% sucrose, 0.02% polysorbate 80, pH 7.5) was administered intravenously via tail vein injection into four C57BL / 6 mice (64-65 days old). For scp776 administration, mice were slightly anesthetized with isoflurane. Blood glucose was measured at baseline and then monitored up to 8 hours after scp776 administration. At all time points, blood glucose measurements were performed without anesthesia using tail blood samples and a portable strip glucometer. Mice were allowed to eat and drink ad libitum during treatment.

[0339] The treatment was then repeated 3 weeks later (t2) on the same group of 4 C57BL / 6 mice (86 days old).

[0340] Table 1: Animal overview [Table 2]

[0341] result:

[0342] Despite frequent handling, SCP776 administered intravenously at 60 mg / kg was well tolerated in all mice, with no abnormal findings. At this dose level, SCP776 induced a significant (but variable) decrease in blood glucose levels. A similar decrease in blood glucose was measured in a second run of treatment.

[0343] Table 2: Baseline glucose [Table 3]

[0344] Table 3: Blood glucose values [Table 4]

[0345] Figure 7 shows the blood glucose curve. scp776 administered iv at a dose of 60 mg / kg significantly reduced blood glucose in C57BL / 6 mice. Results vary, but the mean nadir of the response occurs 3 hours after scp776 administration. The duration of the response is related to the clearance rate (t 1 / 2 This is surprising given that the mean blood glucose levels are <1.5 hours (<1.5 hours). A potential glucose supplementation strategy would aim to maintain blood glucose levels above 100 mg / dL for the first 8 hours.

[0346] Example 2: Cerebroprotective effect and safety of scp776, an IGF-1 fusion protein targeted to damaged tissue, in patients with large vessel ischemic stroke undergoing thrombectomy without thrombolysis scp776 is a targeted insulin-like growth factor 1 (IGF-1) fusion protein. In some embodiments, the targeted insulin-like growth factor 1 (IGF-1) fusion protein is used to inhibit apoptosis and limit cell death due to hypoxia and reperfusion injury in acute ischemic stroke (AIS).

[0347] Objective: ARPEGGIO evaluates the cerebroprotective potential of scp776 in subjects undergoing endovascular thrombectomy (EVT) for AIS.

[0348] DESIGN:ARPEGGIO is a two-part, phase 2, randomized, double-blind, placebo-controlled study in subjects with AIS, enrolling approximately 40 subjects in dose escalation (Part A) and approximately 40 subjects in dose expansion (Part B).

[0349] Population: The main inclusion criteria were AIS within 16 hours, confirmed signs of intracranial occlusion in the intracranial carotid artery and / or M1 middle cerebral artery, and NIHSS ≥ 6, while the main exclusion criteria were acute intracerebral hemorrhage, poor / non-existent collateral circulation, ASPECT score of 0-4, and current IV thrombolytic therapy.

[0350] In Part A, subjects were assigned 1:1:2 overall to Cohort 1 (1.9 mg / kg scp776), Cohort 2 (3.8 mg / kg scp776), or saline placebo. Doses of scp776 or saline placebo were administered IV once daily, 24 hours apart. Subjects will undergo glucose monitoring and dextrose supplementation for at least 48 hours. Safety data collected in Part A of ARPEGGIO will inform the dose selected for expansion in Part B.

[0351] In some embodiments, the IGF-1 activator domain in scp776 inhibits apoptosis and promotes brain protection; the central human serum albumin scaffold extends the half-life of the chimeric protein, and the Annexin V targeting domain binds to phosphatidylserine on the surface of apoptotic cells.

[0352] In a non-human primate model of AIS, treatment with scp776 resulted in a reduction in lesion size, improved neurological function, and a significant survival benefit. scp776 was safe and well tolerated in two phase 1 clinical trials.

[0353] Preclinical efficacy in non-human primate models of AIS: Improved BBB integrity during the acute phase >30% reduction in lesion size in 72 hours Improved neurological function A five-fold reduction in mortality in the SCP-776 treatment group

[0354] In some embodiments, the chimeric proteins provided herein (e.g., scp776) do not bind to IGF-binding proteins (IGFBPs) and are targeted to damaged cells at the site of injury. In some embodiments, effectively targeting damaged cells limits side effects such as hypoglycemia, tonsillar hypertrophy, and allergic reactions. In some embodiments, the chimeric proteins provided herein are efficiently delivered and reduce damage as paracrine pro-survival factors. In some embodiments, the chimeric proteins provided herein (e.g., scp776) are engineered to send pro-survival driving signals to damaged cells.

[0355] In some embodiments, the chimeric proteins provided herein (eg, scp776) are administered intravenously and have a circulating half-life of about 10 hours in healthy subjects.

[0356] In some embodiments, targeted intervention during acute injury may alter the density of injured cells and protect tissue function. After acute injury, cells in the injury center enter apoptosis and spread to surrounding cells. Without intervention, the apoptotic center spreads to the periphery, further impairing tissue function. SCP776 limits the damage caused by acute injury by promoting apoptotic escape in surviving tissue and prevents the injury center from expanding. See Figure 1, which illustrates injury progression at the tissue level. After injury, injured cells and surrounding areas enter the apoptotic pathway. If untreated, this spreads, eventually resulting in a mass of dead cells covered by scar tissue. The size of this scar determines the functional impact on the tissue. Scarring often results in irreversible functional impairment. SCP776 can slow the spread of injury, convert injured cells to healthy ones, and ultimately reduce the size of the resulting scar—thereby protecting tissue function.

[0357] Figures 2A-2B show that scp776 inhibits the apoptotic pathway and promotes escape from apoptosis more effectively than natural growth factors. scp776 strongly interacts with phosphatidylserine (PS), which is translocated during apoptosis, and activates pro-survival IGF-1R signaling in damaged cells at >100-fold lower concentrations.

[0358] Phase 1A

[0359] A single ascending dose of scp776 was first conducted in a human Phase 1A study in healthy volunteers to evaluate its safety, tolerability, and pharmacokinetic (PK) profile in humans. Twenty-four healthy adult male subjects were enrolled in three cohorts (6 active:2 placebo per cohort).

[0360] Serum concentration curves at the three dose levels investigated in this study are shown. In healthy human participants, the circulating half-life of SCP776 is 10.6±1.4 hours. See Figure 3. The results indicate that SCP776 is safe and well tolerated in healthy human subjects.

[0361] Phase 1B

[0362] The Phase 1B study was a randomized, double-blind, placebo-controlled study of the safety and PK of single ascending and multiple IV doses of scp776 in healthy adults receiving continuous infusion of dextrose. The study enrolled 44 healthy adult male and female subjects in six cohorts.

[0363] Supplemental dextrose infusion was implemented to limit the known blood glucose (BG)-lowering effects of IGF1. Comparison of the area above euglycemia (70 mg / dL) in Phase 1A and Phase 1B is shown. Supplemental dextrose in Phase 1B significantly increased the area of ​​euglycemia and maintained BG levels after scp776 administration. See Figure 4. Bedside BG measurements were performed 24 hours after scp776 administration. The area of ​​euglycemia was calculated as the area under the BG curve above 70 mg / dL. The mean euglycemic area was significantly higher with dextrose supplementation (p=0.0041).

[0364] Additional results indicate that a multiple-dose regimen of scp776 is safe and well tolerated when administered with a continuous infusion of supplemental dextrose.

[0365] Preclinical efficacy of scp776 in NHP models of AIS

[0366] SCP776 was tested in a double-blind, non-human primate (Macaca fascicularis) transient middle cerebral artery occlusion model of AIS. Two clips were placed over a 4-hour ischemic period, creating a large infarct and a small penumbra.

[0367] scp776 (n=10) or placebo (n=12) was administered 30 minutes before reperfusion. All animals received a continuous supply of supplemental dextrose via an intravenous infusion pump. Dextrose in water (D10W) was infused at 0.5 mL / kg / hr. The infusion began 30 minutes before the intravenous administration of the first dose of scp776 or placebo. The supplemental dextrose infusion continued at this rate for 120 hours after the first dose.

[0368] Outcomes included MRI imaging, neurological function, and survival.

[0369] Figures 5A and 5B show the preclinical efficacy of scp776 in an NHP model of AIS. Figure 5A: Study PR0362-1 compared three groups: placebo (n = 12), low-dose (24 mg / kg total scp776; n = 10), and high-dose (32 mg / kg total scp776; n = 10). Dose-dependence was observed for all measured parameters. For simplicity, only the high dose is shown. Blood-brain barrier integrity, lesion size, and neuropathy scores are shown as group means ± SEM for the high-dose and placebo groups. Multiple comparisons of means (ANOVA with least significant difference tests) were performed for all three groups to determine significant effects. Values ​​for monkeys that died before scheduled evaluations were considered the maximum observed values. Figure 5B: Survival curves for the high-dose scp776 (n = 10) and placebo (n = 12) groups from Study PR0362-1 are plotted.

[0370] Phase 2

[0371] Study Design: ARPEGGIO is a Phase 2, randomized, placebo-controlled, double-blind study with two parts: sequentially escalating doses in Part A, followed by dose expansion in Part B.

[0372] In Part A, approximately 40 evaluable subjects will be assigned 1:1:2 overall to Cohort 1, Cohort 2, or placebo. Doses of scp776 will be tested sequentially in two cohorts, each with a dose-matched placebo, randomized 1:1 within each cohort, maintaining an overall 1:1:2 ratio. Subjects will receive two doses of either saline (placebo) or scp776, approximately 24 hours apart. In Part B, approximately 40 subjects will be assigned 1:1 to the best therapeutic dose of Part A or placebo.

[0373] On Day 1, subjects who meet eligibility criteria will begin receiving study medication at a stroke center with on-site endovascular facilities. Brain imaging scans and Alberta Stroke Program Early CT (ASPECT) score eligibility confirmation must be performed within 4 hours before the planned EVT. Study medication is intended to be administered within 1 hour of the baseline / qualification scan, if possible, but within 4 hours after the qualification scan. Study medication should be administered before the first pass of the EVT device, if possible, but before resumption of vascular flow. Preferably, at least 5 minutes should elapse between the end of study medication administration and the patency of the vessels for the EVT procedure. Study medication is administered via slow IV infusion over 2 minutes. The second dose of study medication is administered 24 hours after the first dose. Subjects remain hospitalized for at least 48 hours, with data collected through Day 7 if still hospitalized. Subjects return for follow-up visits on Days 30 and 90.

[0374] Due to alterations in the insulin-like growth factor signaling arm, scp776 may lower blood glucose levels. Prior to administration of the study drug (e.g., 1 to 120 minutes), all subjects began an IV infusion of 10% dextrose in water (D10) at a rate of between 0.1 and 2.0 mL / kg / h. Dextrose administration may continue for at least 48 hours (e.g., about 48 to about 120 hours, about 48 to about 72 hours, about 48 to about 96 hours, about 48 to about 120 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours) and was administered according to a blood glucose management plan (BGMP) protocol. See Figure 6.

[0375] Inclusion Criteria: · 18 years of age or older. Acute ischemic stroke (AIS) where immediate endovascular treatment is intended. Disabling stroke defined as a baseline National Institutes of Health Stroke Score (NIHSS) equal to or greater than 6 at the time of randomization. -Confirmed evidence of intracranial occlusion in one or more of the following locations based on imaging: intracranial carotid artery and / or M1 middle cerebral artery (MCA).Time from AIS onset (last known healthy state) to randomization time is <16 hours. Before the onset of AIS (24 hours before the onset of stroke), participants must be independently functional in activities of daily living, with a modified Rankin Score of 0 or 1. Participants must live in their own home, apartment, or senior lodge without requiring nursing care.

[0376] Exclusion criteria: Evidence of acute intracerebral hemorrhage on imaging confirmation according to the Radiology Laboratory Manual. Poor / non-existent collateral circulation (e.g., collateral score of 0 or 1). ·ASPECT score of 0-4. Current AIS treated with IV thrombolytic therapy or subject had received thrombolytic therapy within the last 24 hours. · Intentional use of any intravascular thrombectomy device that is not FDA approved. · The use of intra-arterial thrombolysis is planned. Clinical history, previous imaging, or clinical diagnosis suggests a chronic intracranial obstruction or suspected intracranial dissection, predicting that endovascular intervention is unlikely to be successful.

[0377] Table 4: Objectives and associated endpoints [Table 5]

[0378] Example 3: Effect of starting dextrose infusion rate on the total dextrose required to support euglycemia in healthy human subjects receiving intravenous infusion of scp776 The objective of this study was to identify dextrose supplementation parameters that facilitate maintaining normoglycemia with minimal total dextrose infusion volume.

[0379] scp776 is a tripartite fusion protein containing a modified IGF-1 signaling arm that targets damaged cells, promotes apoptosis escape, and maintains healthy tissue. By targeting damaged cells, scp776 can be administered at higher doses than IGF-1; however, the glucose-lowering effect of scp776 has been observed in both preclinical toxicity studies and first-in-human single-ascending-dose clinical trials. A strategy of supplemental dextrose infusion and responsive glucose monitoring after scp776 administration has been developed in multiple-ascending-dose studies in healthy male and female participants. The blood glucose management strategy described herein can be deployed in acute ischemic stroke patients undergoing thrombectomy.

[0380] IGF-1-based therapies can lower blood glucose: Insulin-like growth factor-1 (IGF-1) is structurally related to insulin and has widespread receptor binding throughout the body. IGF-1 is a hormone of clinical interest due to a wide range of important physiological actions, including neonatal growth and development, inhibition of apoptosis, and a role in glucose regulation, with primary targeting of postnatal skeletal muscle and bone growth. IGF-1 has been evaluated for therapeutically lowering glucose levels in diabetic patients; however, its therapeutic potential has been limited by the occurrence of undesirable side effects in this population, including jaw pain, joint pain, and tachycardia, while its significant effects on glucose metabolism and ability to induce hypoglycemia have limited its clinical development in other indications.

[0381] Figure 9 shows that the unique pharmacological and systemic effects of the IGF-1 component of scp776 can be addressed by dextrose supplementation.

[0382] Glycemic Control in Stroke: Hyperglycemia is common in the setting of acute ischemic stroke (AIS), both due to underlying diabetes and as a stress response to stroke in patients without diabetes. Conversely, if a long time has passed since last known well (LKW), stroke patients may be hypoglycemic due to prolonged fasting. Although hyperglycemia is a concern, trials using intensive glucose management with strict insulin control have not shown improved functional outcomes or mortality, but instead have shown increased morbidity due to hypoglycemic events. New therapies for the treatment of acute stroke must consider the comorbidity of hyperglycemia in these patients and the confounding effects of these compounds while following AHA guidance for glucose management in AIS.

[0383] Phase 1: Dextrose infusion strategy

[0384] Figure 10 shows the Phase 1a single ascending dose (SAD) study and the Phase 1b multiple ascending dose (MAD) study. The Phase 1a SAD study enrolled only men. The Phase 1b MAD study enrolled men and women. Blood glucose levels were monitored at 30-minute intervals. Scheduled meals included: breakfast (8:00 AM), lunch (12:00 PM), dinner (5:00 PM), and a midnight snack (8:00 PM).

[0385] In Phase 1a trials, blood glucose lowering was addressed with oral glucose (eg, juice).

[0386] In the phase 1b trial, IV dextrose was supplemented and changes in blood glucose were managed by adjusting the infusion rate.

[0387] In the Phase 1B study SCP-CL-0002, healthy male and female subjects received supplemental IV dextrose prior to administration. Individual subjects' blood glucose levels responded to changes in the dextrose infusion rate within standard monitoring intervals (i.e., 0.5 hours). Although some subjects responded more quickly than others, IV dextrose supplementation successfully reversed hypoglycemic episodes. While ongoing, it was recognized that a high infusion rate (i.e., 6 mL / kg / hr) may have perpetuated the need for supplementation. It is noteworthy that the decline in blood glucose occurred shortly after the Tmax of SCP776 (~0.5 hours) and stabilized as serum SCP776 concentrations declined and dextrose supplementation was optimized. Dose reduction, combined with adjustments to the slow-start dextrose infusion rate and rate of increase, minimized the total infusion volume while controlling blood glucose. These dose levels and dextrose supplementation strategies are feasible in standard clinical situations when administering SCP776 to patients. See Figure 8.

[0388] In cohorts 1-3, the dextrose infusion rate was initiated at a high rate with large rate adjustments. In cohorts 4-6, the dextrose supplementation strategy was initiated at a low rate immediately prior to scp776 and adjusted to smaller incremental rate adjustments in response to glucose monitoring. As shown, significantly less supplemental dextrose was required to maintain euglycemia in cohorts 4-6.

[0389] In cohorts 1, 2, and 3, subjects received scp776 at total doses of 4 mg / kg, 4 mg / kg, and 6 mg / kg, respectively. These subjects were started on a 5% dextrose in saline (D5NS) or 10% dextrose in saline (D10W) infusion rate of 1 mL / kg / h to 2 mL / kg / h 2 hours prior to the maximum administration of scp776. In these subjects, the dextrose infusion rate was adjusted in large increments (i.e., +1 mL / kg / h to +2 mL / kg / h) in response to a measured blood glucose decline of ≥ 5 mg / dL.

[0390] In cohorts 4, 5, and 6, subjects received scp776 at total doses of 6 mg / kg, 7.5 mg / kg, and 6.2 mg / kg, respectively. These subjects were started on a D10W infusion rate within 30 minutes prior to administration of scp776. In these subjects, the dextrose infusion rate was adjusted in small increments (i.e., +0.1 mL / kg / hr to +0.5 mL / kg / hr) in response to a measured blood glucose decline of ≥5 mg / dL.

[0391] Supplemental dextrose reduces the frequency of hypoglycemic events

[0392] In the first human trial, SCP-CL-0001, subjects received 2 or 4 mg / kg of SCP776 without constant dextrose supplementation. At the same dose levels with IV dextrose supplementation in SCP-CL-0002, subjects experienced fewer hypoglycemic events and better maintained normal blood glucose levels for a 6-hour period following administration. For reference, hypoglycemic events in the saline placebo group are also shown. (Note: Meals were scheduled at t=0 & 4 hours.) See Figures 11A-11C.

[0393] In all subjects receiving scp776 with supplemental IV dextrose, no grade 2 or higher hypoglycemic events (CTCAE criteria of ≦54 mg / dL) were observed in the post-dose period, and normoglycemia was maintained with minimal deviations to grade 1 hypoglycemia (CTCAE criteria of ≦75 mg / dL).

[0394] FIG. 8 shows the mean total amount of supplemental dextrose solution infused for subjects in each of Cohorts 1-6.

[0395] Cohort 1 received one dose of scp776 at 4 mg / kg. Cohort 2 received one dose of scp776 at 4 mg / kg. Cohort 3 received two doses of scp776 at 3 mg / kg on days 1 and 3. Cohort 4 received three doses of scp776 at 2 mg / kg on days 1, 2, and 3. Cohort 5 received two doses of scp776 at 2 mg / kg on days 1 and 2, a dose of scp776 at 1.75 mg / kg on day 3, and a dose of scp776 at 1.5 mg / kg on day 4. Cohort 6 received scp776 at 2 mg / kg on day 1, 1.8 mg / kg on day 2, 1.4 mg / kg on day 3, and 1 mg / kg on day 4. Subjects in cohorts 1, 2, or 3 received, on average, approximately 7,000 mL of dextrose supplement solution in the first 48 hours. Subjects in cohorts 4, 5, or 6 received, on average, approximately 1,000 mL of dextrose supplement solution in the first 48 hours.

[0396] In support of the three-dose regimen in Cohort 4, subjects received, on average, approximately 1,600 mL of dextrose supplementation solution 96 hours after the first scp776 dose.

[0397] In support of the four-dose regimen in cohorts 5 and 6, subjects received, on average, approximately 2,000 mL of dextrose supplementation solution 120 hours after the first scp776 dose.

[0398] The significant difference in the total amount of dextrose supplement solution required to support euglycemia in cohorts 1-3 compared to cohorts 4-6 contributes to modifications in the blood glucose management program.

[0399] Because hypoglycemia is a known side effect of SCP776, all subjects in this study received supplemental dextrose via controlled IV infusion. The supplemental dextrose infusion was implemented to minimize potential risks associated with sustained low blood glucose levels. The supplemental dextrose solution and route of administration were chosen to mimic the clinical situation of patients who would eventually become ill and receive SCP776.

[0400] An adaptive approach to supplemental dextrose infusion was used in the study series. In general, subjects did better when the dextrose infusion was initiated immediately prior to the first dose rather than 30 minutes or more before the dose. Initial dextrose infusion parameters (e.g., initiation rate, dextrose solution) were consistent within subjects in a given dose group. As the study progressed, initial parameters were modified based on blood glucose control in previous cohorts and adjustments to the SCP776 dosing regimen. Initial dextrose infusion parameters are summarized below by cohort and / or dose group.

[0401] In Cohort 1, subjects received a continuous infusion of 5% dextrose in saline (D5NS) at a rate of 0.5 mL / kg / hr immediately prior to the administration of scp776 or placebo, continuing for the first 48 hours after administration. However, due to the tendency for blood glucose levels to drop sharply, necessitating an increase in the amount of dextrose, it was determined that the large doses of D5NS required to alleviate low blood glucose levels were inappropriate or unmanageable for all subjects. In subjects for whom D5NS was inappropriate, a similar strategy of dextrose supplementation (e.g., 10% dextrose solution in sterile water (D10W)) was implemented. Given this, the single dose of 4 mg / kg administered in Cohort 1 was repeated in Cohort 2, with the initial supplemental dextrose infusion modified to D10W at a starting rate of 2 mL / kg / hr.

[0402] In cohorts 2 through 4, subjects received a continuous infusion of D10W at an initial rate of 2 mL / kg / hr or less before administration of scp776 or placebo, and continued for the first 48 hours after the last dose (i.e., 96 hours after the first dose for cohorts 3 and 4). Additionally, in the first group of subjects receiving cohort 2, the dextrose infusion was initiated approximately 1 hour before administration of scp776 or placebo; this was thought to have a priming effect on endogenous insulin. Therefore, in subsequent treatment groups and cohorts, the start of the dextrose infusion was moved back to immediately before administration of scp776 or placebo. Although the downward trend in blood glucose levels was attenuated (i.e., the trend improved) compared with the initial steep downward trend in Cohort 1, significant adjustments in the D10W infusion rate were still required to alleviate the low blood glucose levels in most subjects in Cohort 2 (single dose of 4 mg / kg scp776 or placebo) and the first four subjects in Cohort 3 (two QOD doses of 3 mg / kg scp776 or placebo). In the remaining subjects receiving treatment in Cohort 3, the initial supplemental dextrose (D10W) infusion rate was reduced to 0.5 mL / kg / hr, and small adjustments in the D10W infusion rate were used as blood glucose levels decreased. This allowed for improved blood glucose control with smaller doses of supplemental dextrose.

[0403] By the start of Cohort 4, the initial supplemental D10W infusion rate was low, requiring small / fine adjustments in the D10W infusion rate to alleviate low blood glucose levels. In Cohorts 5 and 6, subjects received a continuous infusion of D10W at an initial rate of 0.1 mL / kg / hr immediately prior to administration of scp776 or placebo. The dextrose infusion could then be adjusted, paused, or discontinued in Cohort 5 and 6 subjects at any time after administration of the first dose, according to the Blood Glucose Management Plan (BGMP, Table 5 and Example 4) protocol, with small incremental changes in the infusion rate based on bedside glucose monitoring.

[0404] Table 5. Dextrose Infusion Strategy Guidelines for the Management of Euglycemia [Table 6] Abbreviations: BG = blood glucose; BGMP = blood glucose management plan; D10 = 10% dextrose in water

[0405] Based on our experience in the first-in-human trial of scp776 (SCP-CL-0001), a strict blood glucose management regimen (BGMP) was implemented in the Phase 1b MAD trial (SCP-CL-0002). Continuous glucose monitoring devices and bedside point-of-care measurements were used to monitor BG levels in all subjects, and subjects received supplemental IV dextrose at infusion rates adjusted to maintain euglycemia.

[0406] The experience in both phase 1 trials influenced the design of the BGMP currently being deployed in a phase 2 trial of scp776 in patients with acute ischemic stroke.

[0407] In the setting of stroke, the timing of the most recent meal and baseline blood glucose levels will vary.

[0408] Reductions in blood glucose are most likely to occur within the first 6 hours after administration of SCP776 and in subjects with low baseline levels.

[0409] A slow supplemental dextrose infusion should be initiated immediately prior to administration of SCP776.

[0410] Sensitive incremental adjustments in the dextrose infusion rate are more effective than delayed or large rate adjustments.

[0411] · Oral intake of a balanced diet should be encouraged.

[0412] Example 4: Blood glucose control plan A subset of subjects receiving scp776 study drug may experience hypoglycemia, which is managed with a combination of frequent glucose testing and supplemental dextrose.

[0413] At all times, clinical judgment should be used for glucose management, and BGMPs should not interfere with the institution's standard clinical care.

[0414] In some embodiments, protocol-specified blood glucose control continues for 48 hours after the first study drug administration, after which patients may continue dextrose supplementation and blood glucose monitoring according to standard of care.

[0415] Glucose Monitoring and Management All subjects receiving study medication will undergo a minimum of 48 hours of blood glucose (BG) monitoring under the following guidelines:

[0416] BG levels are used to determine the rate of dextrose supplementation based on Table 5.

[0417] After each dose of study drug, all subjects will undergo blood glucose testing at 30 minutes (±15 minutes), and then hourly (±20 minutes) until 8 hours after dosing. After 8 hours, if blood glucose levels stabilize, glucose testing may change to every 2 hours (±20 minutes). A stable blood glucose level is defined as a blood glucose >100 mg / dL for 4 consecutive hours without the need to increase the rate of dextrose infusion.

[0418] Any change in clinical status suggesting a decrease in blood glucose (e.g., change in mental status after administration) requires immediate glucose testing and, if no longer stable, a return to hourly testing and adjustment of the dextrose rate until stable.

[0419] If your blood glucose levels were stable but are no longer stable: If BG < 80 mg / dL, recheck BG every 15 minutes and adjust / administer dextrose according to Table 5 until BG is confirmed to be > 80 mg / dL. When BG > 80 mg / dose is confirmed, return to hourly glucose testing within 8 hours of administration. If more than 8 hours post-dose, return to hourly glucose testing until BG is stable (BG > 100 mg / dL for 4 consecutive hours without increasing dextrose administration), then glucose testing may be changed again to every 2 hours.

[0420] BG can be performed using a central laboratory test, a bedside test (e.g., i-STAT (登録商標) blood gases), or by fingerstick.

[0421] Dextrose Infusion Prior to administration of study medication, all subjects will begin an IV infusion of D10 starting at a rate of 0.1 mL / kg / h.

[0422] The dextrose infusion should be started no more than 30 minutes before the study drug injection.

[0423] Dextrose administration is continued for at least 48 hours if BG levels are not >150 mg / dL on two consecutive readings ≥30 minutes apart, in which case D10 can be discontinued or slowed down.

[0424] If BG levels are falling or are <80 mg / dL, increase the D10 rate.

[0425] If there is clinical concern about administering excessive free water, D10 may be switched to 5% dextrose in saline at the discretion of the site's clinical trial team. Any changes in dextrose infusion rate should be recorded on the case report form.

[0426] If the rate of decline in BG levels is deemed to be rapid, the clinical team may deviate from the protocol in Table 5 and should document the reason for the change from the plan.

[0427] If a 50% dextrose infusion is given for any reason, BG testing (fingerstick or laboratory glucose) should be performed every 15 minutes until the BG level is >80 mg / dL.

[0428] If the rate of D10 infusion exceeds 1 mL / kg / h, the medical monitor for the study should be contacted.

[0429] Specific examples of compositions, methods, and kits are described herein for illustrative purposes. These are examples only. The techniques provided herein may be applied to systems other than those of the examples described above. Many changes, modifications, additions, omissions, and substitutions are possible in practicing the invention. The present disclosure includes variations of the described embodiments that will be apparent to those of skill in the art, including variations obtained by: substituting features, elements, and / or acts with equivalent features, elements, and / or acts; mixing and matching features, elements, and / or acts from different embodiments; combining features, elements, and / or acts from the embodiments described herein with features, elements, and / or acts from other technologies; and / or omitting combinations of features, elements, and / or acts from the described embodiments.

[0430] Incorporation by Reference All publications, patents, and sequence database entries mentioned herein are herein incorporated by reference in their entirety, to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

Claims

1. 1. A method of treating a subject in need thereof with an IGF-1 chimeric protein, the method comprising: administering a solution comprising an effective amount of dextrose prior to administering the IGF-1 chimeric protein, wherein the administration is over a period of about 48 hours; and administering an effective amount of a pharmaceutical composition comprising an IGF-1 chimeric protein; wherein the effective amount of dextrose supports normoglycemia.

2. 10. The method of claim 1, wherein the solution containing an effective amount of dextrose is administered by infusion to a subject in need thereof.

3. 3. The method of claim 2, comprising administering a solution containing an effective amount of dextrose at a starting infusion rate of about 0.1 mL / kg / h.

4. 4. The method of claim 3, further comprising adjusting the infusion rate in increments of +0.05 mL / kg / hr to +2 mL / kg / hr.

5. 3. The method of claim 1 or claim 2, wherein the solution containing an effective amount of dextrose comprises about 5% to about 10% dextrose (w / v) in water or saline solution.

6. 3. The method of claim 1 or claim 2, wherein the solution comprising an effective amount of dextrose is administered about 1 minute to about 30 minutes prior to administration of the IGF-1 chimeric protein.

7. 3. The method of claim 1 or claim 2, wherein the IGF-1 chimeric protein comprises a targeting domain comprising human annexin 5 (AnxV) or a variant thereof, and an activator domain comprising insulin-like growth factor 1 (IGF-1) or a variant thereof.

8. 8. The method of claim 7, wherein the IGF-1 chimeric protein further comprises a peptide, wherein the peptide extends the half-life of the IGF-1 chimeric protein.

9. The method of claim 7, wherein the targeting domain is a non-internalizing variant of annexin 5.

10. 8. The method of claim 7, wherein the IGF-1 chimeric protein is not substantially internalized by the cell.

11. 8. The method of claim 7, wherein the IGF-I chimeric protein comprises a non-internalizing variant of annexin 5, wherein the non-internalizing variant of annexin 5 comprises one or more mutations, wherein the one or more mutations comprise a substitution at a position corresponding to C316 and, optionally, one or more positions corresponding to R63, K70, K101, E138, D139, N160, and combinations thereof.

12. 8. The method of claim 7, wherein the activator domain of the IGF-1 chimeric protein is a variant of human insulin-like growth factor IGF-1 comprising one or more mutations, wherein the one or more mutations consist of substitutions at one or more positions corresponding to E3, Y24, Y31, Y60, and combinations thereof.

13. 8. The method of claim 7, wherein the IGF-I chimeric protein further comprises a half-life modulator comprising a variant of human serum albumin (HSA) comprising one or more mutations, wherein the one or more mutations consist of substitutions at one or more positions corresponding to C58 and N527, and combinations thereof.

14. 8. The method of claim 7, wherein the IGF-1 chimeric protein comprises or consists of IGF1(E3R / Y31A)_lk7_HSA26-609(C58S / N527Q)_lk7_AnxV2-320(R63A / K70A / K101A / E138A / D139G / N160A / C316A).

15. 8. The method of claim 7, wherein the IGF-1 chimeric protein comprises or consists of the amino acid sequence set forth in SEQ ID NO:

24.

16. 3. The method of claim 1 or claim 2, comprising administering an effective amount of an IGF-1 chimeric protein in a tapered manner over a period of 2 to 14 days or longer.

17. 3. The method of claim 1 or claim 2, wherein administering an effective amount of an IGF-1 chimeric protein inhibits apoptosis.

18. 3. The method of claim 1 or claim 2, wherein the pharmaceutical composition further comprises at least one physiologically acceptable carrier.

19. 3. The method of claim 1 or claim 2, wherein the subject in need thereof is a human.

20. 20. The method of claim 19, wherein the subject in need thereof has an acute CNS disorder.

21. 20. The method of claim 19, wherein the subject in need thereof has an acute cardiovascular disorder.

22. 20. The method of claim 19, wherein the subject in need thereof has acute radiation syndrome.

23. 20. The method of claim 19, wherein the subject in need thereof has a chemo-inhalation injury.

24. 20. The method of claim 19, wherein the subject in need thereof has a dermatological disorder.

25. 20. The method of claim 19, wherein the subject in need thereof has a traumatic injury.

26. 20. The method of claim 19, wherein the subject in need thereof has a chronic neurodegenerative disease.

27. 20. The method of claim 19, wherein the subject in need thereof is undergoing a procedure that puts the subject at risk of iatrogenic injury.

28. 20. The method of claim 19, wherein the subject in need thereof is undergoing organ transplantation treatment.

29. 20. The method of claim 19, wherein the subject in need thereof is undergoing a cosmetic dermatology treatment.

30. 1. An IGF-1 chimeric protein for use in a method of treating a subject in need thereof, the method comprising: administering a solution comprising an effective amount of dextrose prior to administering the IGF-1 chimeric protein, wherein the administration is over a period of about 48 hours; and administering an effective amount of a pharmaceutical composition comprising an IGF-1 chimeric protein; Including, An IGF-1 chimeric protein in which an effective amount of dextrose supports normoglycemia.

31. 1. Dextrose for use in a method of treating a subject in need thereof with an IGF-I chimeric protein, the method comprising: administering an effective amount of a solution containing said dextrose prior to administering an IGF-1 chimeric protein, wherein the administration is over a period of about 48 hours; and administering an effective amount of a pharmaceutical composition comprising an IGF-1 chimeric protein; Including, Dextrose, an effective amount of dextrose supports normoglycemia.

32. 1. A combination of an IGF-1 chimeric protein and dextrose for use in a method of treating a subject in need thereof, the method comprising: administering a solution comprising an effective amount of dextrose prior to administering the IGF-1 chimeric protein, wherein the administration is over a period of about 48 hours; and administering an effective amount of a pharmaceutical composition comprising an IGF-1 chimeric protein; Including, A combination in which an effective amount of dextrose supports normoglycemia.

33. 1. Use of an IGF-1 chimeric protein in the manufacture of a medicament for treating a subject in need thereof, wherein a solution comprising an effective amount of dextrose is administered prior to administering a pharmaceutical composition comprising an effective amount of an IGF-1 chimeric protein, wherein the administration of the solution is over a period of about 48 hours, and wherein the effective amount of dextrose supports normoglycemia.

34. 1. Use of dextrose and an IGF-1 chimeric protein in the manufacture of a medicament for treating a subject in need thereof, wherein a solution comprising an effective amount of dextrose is administered prior to administering a pharmaceutical composition comprising an effective amount of an IGF-1 chimeric protein, and wherein the administration of the solution comprising the effective amount of dextrose is for a period of about 48 hours, and wherein the effective amount of dextrose supports normoglycemia.

35. 1. Use of a combination of an IGF-1 chimeric protein and dextrose in the manufacture of a medicament for treating a subject in need thereof, wherein a solution comprising an effective amount of dextrose is administered prior to administering a pharmaceutical composition comprising an effective amount of an IGF-1 chimeric protein, and wherein the administration of the solution comprising the effective amount of dextrose is for a period of about 48 hours, and wherein the effective amount of dextrose supports normoglycemia.

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