HEMATOPOIESIS, AND STIMULATE BONE GROWTH

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application 62/587,784 filed on November 17, 2017 and U.S. Provisional Application 62/625,242 filed on February 1, 2018, both herein incorporated by reference in their entireties.

FIELD OF THE DISCLOSURE

Provided herein are methods of using copeptin to modulate the immune response and hematopoiesis, such as erythropoiesis, lymphopoiesis, platelet formation, or any combination thereof, and stimulate bone growth and fracture healing. Such methods can be used to treat hematopoietic disorders, such as anemia and polycythemia, autoimmune disorders, such as encephalomyelitis and diabetes, and bone disorders, such as osteopenia and osteoporosis. Also provided are compositions that include copeptin and one or more additional therapeutic molecules, for use with the disclosed methods.

BACKGROUND

Copeptin, an arginine vasopressin (AVP)-associated glycopeptide, was first described by Holwerda in 1972. It is derived from the cleavage of the precursor of AVP, produced in an equimolar ratio, that is, 1: 1 ratio, in the hypothalamus/posterior pituitary. AVP has a short half-life of 5-20 minutes, making it difficult to measure. Unlike AVP, copeptin is stable for days at room temperature and can easily be measured. For this reason, copeptin is used as a surrogate to measure the levels of AVP in patients. In spite of being used to measure AVP, the exact function of copeptin remained unknown.

Anemia is a condition in which the blood is either low in total volume or is deficient in red blood cells or hemoglobin. Anemia can occur for several reasons including hemorrhage and following chemotherapy. Erythropoietin stimulating agents are commonly administered to stimulate red blood cell production and thus, treat anemia. However, these agents can be ineffective at treating anemia because they are slow to act, are ineffective in iron deficient subjects, and can cause high blood pressure thereby increasing risk of death, heart failure, heart attack and stroke. Therefore, a need exists for methods of modulating erythropoiesis in anemic subjects, which are quicker to act, and not associated with the aforementioned side effects.

Polycythemia is a condition associated with a pathological increase in red blood cell production.

An autoimmune disease is a disease associated with a dysfunctional immune system, where the body becomes the target of its own immune system.

Osteoporosis is a degenerative disease in which the density and quality of bones are reduced. There is no real cure for these diseases. Therefore, a need exists for methods of modulating hematopoiesis and the immune system, and stimulating bone formation, which are quicker to act, and not associated with common side effects.

SUMMARY

Based on the data provided herein, it is proposed that copeptin participates in

hematopoiesis, such as red blood cell, platelet and white blood cell production, the immune response, such as B and T cell formation, and osteoinduction, such as bone and cartilage formation. Based on these findings, provided herein are methods of modulating (such as increasing or decreasing) hematopoiesis, for example by modulating hematopoietic stem cell (HSC) proliferation (such as increasing or decreasing), erythropoiesis, lymphopoiesis, or combinations thereof. Also provided herein are methods of modulating an immune response, for example by regulating T cell formation, B cell formation or combinations thereof. In addition, provided herein are methods of stimulating bone and cartilage formation, such as osteoblast formation, osteocyte formation, chondrocyte formation, adipocyte formation, or combinations thereof. Such methods can be used to treat or prevent a disease associated with such conditions, such as anemia, hemorrhage, infection, autoimmune disease, polycythemia, and osteoporosis.

Such methods include administering an effective amount of copeptin to a subject in need thereof, thereby modulating HSC proliferation (such as erythropoiesis and/or lymphopoiesis), the immune response (such as B cell and T cell formation), or stimulating bone formation (such as osteoblast, osteocyte, chondrocyte, adipocyte formation, or combinations thereof). Exemplary copeptin peptides are provided in SEQ ID NOS: 1-4 (also see FIG. 1B). The copeptin administered may or may not be glycosylated.

Exemplary subjects that can be treated with the disclosed methods include vertebrates, such as birds, mammals, fish, and reptiles. In some examples, mammals include veterinary animals. In some examples, mammals include humans. In some examples, the subject treated was previously non-responsive or is at risk of having an adverse reaction to erythropoietin (EPO). In some examples, the subject treated has or is at risk of developing a condition associated with anemia, such as anemia due to hemorrhage, hemolysis, or ineffective hematopoiesis. In some examples, the subject treated is receiving, will receive, or previously received chemotherapy and/or radiation therapy, for example for the treatment of a cancer (and in some examples developed anemia or is at risk of anemia, developed or is at risk of infection, as a result of the chemotherapy or radiation therapy). Thus, the disclosed methods can be used to treat or prevent anemia. In some examples, the subject treated has or is at risk of developing a condition associated with polycythemia, such as polycythemia due to a pathological increase in RBC production. Thus, the disclosed methods can be used to treat or prevent polycythemia. In other examples, the subject treated has or is at risk of developing a condition associated with a dysfunctional immune response, such as an autoimmune disease. Thus, the disclosed methods can be used to treat or prevent an autoimmune disease. In some other examples, the subject treated has or is at risk of developing a condition associated with bone mass deficiency, such as osteopenia or osteoporosis due to a pathological increase in osteoclast production. Thus, the disclosed methods can be used to treat or prevent osteopenia and osteoporosis.

The disclosed methods can include administering one or more additional therapeutic agents, such as an erythropoiesis stimulatory molecule (e.g., EPO, nicotine, AVP, an AVPR1B receptor agonist (such as, but not limited to, d[Cha ⁴ ]AVP; d[Cha ⁴ , Lys ⁸ ]VP, d[Leu ⁴ ]AVP, d[Leu ⁴ ,Lys ⁸ ]VP, or other small molecule agonist), or combinations thereof). The additional therapeutic agents can be administered before, during or after administration of the copeptin.

Also provided are compositions that include copeptin and one or more additional therapeutic agents, such as one or more erythropoiesis modulatory molecules (such as stimulatory molecules), one or more lymphopoiesis modulatory molecules (such as lymphopoiesis stimulatory or inhibitory molecules), one or more autoimmune inhibitory molecules, one or more bone and cartilage stimulatory molecules, or combinations thereof. In some examples, the additional therapeutic agent is one or more of erythropoietin, nicotine, AVP, or an AVPR1B receptor agonist (such as, d[Cha ⁴ ]AVP; d[Cha ⁴ , Lys ⁸ ]VP, d[Leu ⁴ ]AVP, d[Leu ⁴ ,Lys ⁸ ]VP, or other small molecule agonist). Such compositions can also include a physiologically acceptable carrier.

Also provided are methods for treating or preventing polycythemia, for example by administering a therapeutically effective amount of copeptin (or a copeptin antagonist, or an antibody directed against copeptin), alone or with one or more additional therapeutic agents, such as A VP, AVPR1B agonist(s) (such as, d[Cha ⁴ ]AVP; d[Cha ⁴ , Lys ⁸ ]VP, d[Leu ⁴ ]AVP, d[Leu ⁴ ,Lys ⁸ ]VP, or other small molecule agonist), EPO, or any combination thereof.

Also provided herein are methods for modulating erythropoiesis, lymphopoiesis or both in a subject in need thereof, which include administering to the subject an effective amount of copeptin. The subject may have or may be at risk of developing a condition associated with anemia, may be non-responsive to or is at risk of developing an adverse reaction to erythropoietin (EPO), may have or is at risk of developing an infection or a cancer, may be receiving or has received chemotherapy and/or radiation therapy, or may have or may be at risk of developing polycythemia. The methods may further include administering a therapeutically effective amount of one or more additional therapeutic agents, such as one or more of AVP, AVPR1B agonist(s) (such as, d[Cha ⁴ ]AVP;

d[Cha ⁴ , Lys ⁸ ]VP, d[Leu ⁴ ]AVP, d[Leu ⁴ ,Lys ⁸ ]VP, or other small molecule agonist), and EPO.

Also provided herein are methods of modulating an immune response in a subject in need thereof, which includes administering to the subject a therapeutically effective amount of copeptin. The subject may have or may be at risk of developing an autoimmune disease. Modulating an immune response may include regulating T cell formation, B cell formation, or both. The autoimmune disease may be encephalomyelitis (EAE), lupus, type 1 diabetes, rheumatoid arthritis, celiac disease, Sjogren's syndrome, polymyalgia rheumatic, multiple sclerosis, ankylosing spondylitis, alopecia areata, vasculitis or temporal arteritis. These methods may further include administering to the subject a therapeutically effective amount of one or more additional therapeutic agents, such as one or more of AVP, AVPR1B agonist(s) (such as, d[Cha ⁴ ]AVP; d[Cha ⁴ , Lys ⁸ ]VP, d[Leu ⁴ ]AVP, d[Leu ⁴ ,Lys ⁸ ]VP, or other small molecule agonist), and EPO.

Also provided herein are methods of treating diabetes in a subject in need thereof, which comprise administering to the subject a therapeutically effective amount of one or more of AVP, AVPR1B agonist(s) (such as, d[Cha ⁴ ]AVP; d[Cha ⁴ , Lys ⁸ ]VP, d[Leu ⁴ ]AVP, d[Leu ⁴ ,Lys ⁸ ]VP, or other small molecule agonist), and EPO, and a therapeutically effective amount of copeptin. The diabetes may be central diabetes insipidus.

Also provided herein are methods of stimulating bone, cartilage or both in a subject in need thereof, which includes administering to the subject a therapeutically effective amount of copeptin. The subject may have or may be at risk of developing a fracture, or may have or may be at risk of developing osteoporosis. Stimulating bone may comprise increasing bone mass density in the subject. The subject may have, may have had or may be at risk of having knee replacement surgery.

The foregoing and other features of the disclosure will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a schematic drawing showing the pre-pre-vasopressin precursor showing the positon, and size (in amino acids) of AVP, neurophysin II, and copeptin.

FIG. 1B is an alignment of mouse, rat, human, and cow copeptin sequences (SEQ ID NOS: 2, 3, 1 and 4 from top to bottom).

FIG. 2A shows an overview of the methods used to show the effect of copeptin (CP) administration on steady-state erythropoiesis.

FIGS. 2B-2F are bar graphs showing the effect of PBS or copeptin on (B) total BM cellularity, (C) percent cells in the BM that are erythroid, (D) percent of cells in the BM that art myeloid, (E) percent of cells the BM that are B cells, and (F) percent of cells in the BM that are T cells.

FIGS. 3A-3D are bar graphs showing the effect of PBS or copeptin (A) percent cells in the spleen that are erythroid, (B) percent of cells in the spleen that art myeloid, (C) percent of cells the spleen that are B cells, and (D) percent of cells in the spleen that are T cells.

FIGS. 4A-4B show populations of erythroid precursors in mice injected with PBS as compared with copeptin, (A) gating of populations, and (B) bar graph showing percent of erythroid subpopulations observed in the bone marrow cells based on the determination in (A).

FIGS. 5A-5B show analysis of peripheral blood from mice injected with PBS as compared with copeptin, (A) percent of RBCs in the blood, and (B) percent of reticulocytes in the blood.

FIG. 6 is a bar graph showing the effect of PBS or copeptin on different progenitor cells.

FIG. 7 is a schematic overview showing the effect of copeptin at a steady state in the bone marrow and peripheral blood.

FIG. 8 A shows methods used to show the effect of copeptin (CP) or PBS administration on stress erythropoiesis.

FIGS. 8B-8C are bar graphs showing the effect of PBS or copeptin on the cellular populations in the (B) BM and (C) spleen in a PHZ-induced model of anemia. FIG. 9 is a bar graph showing the effect of PBS or copeptin on the reticulocyte count in a PHZ-induced model of anemia.

FIGS. 10A-10B are bar graphs showing the effect of PBS or copeptin on different progenitor cells in (A) bone marrow, or (B) spleen, in a PHZ-induced model of anemia.

SEQUENCE LISTING

The amino acid sequences listed in the accompanying sequence listing are shown using standard three letter code for amino acids, as defined in 37 C.F.R. 1.822. The Sequence Listing is submitted as an ASCII text file, created on November 5, 2018, 8 KB, which is incorporated by reference herein. In the accompanying sequence listing:

SEQ ID NO: 1 is an exemplary human copeptin sequence.

SEQ ID NO: 2 is an exemplary mouse copeptin sequence.

SEQ ID NO: 3 is an exemplary rat copeptin sequence.

SEQ ID NO: 4 is an exemplary cow copeptin sequence.

SEQ ID NO: 5 is an exemplary MANF signal peptide.

SEQ ID NO: 6 is an exemplary CDNF signal peptide.

SEQ ID NO: 7 is an exemplary BIP signal peptide.

SEQ ID NO: 8 is an exemplary gaussia luciferase signal peptide.

SEQ ID NO: 9 is an exemplary albumin signal peptide.

SEQ ID NO: 10 is an exemplary GDNF signal peptide.

SEQ ID NO: 11 is an exemplary BDNF signal peptide.

DETAILED DESCRIPTION

The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein,“comprising” means“including” and the singular forms“a” or“an” or“the” include plural references unless the context clearly dictates otherwise. For example, reference to “comprising a therapeutic agent” includes one or a plurality of such therapeutic agents. The term “or” refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. For example, the phrase“A or B” refers to A, B, or a combination of both A and B. Furthermore, the various elements, features and steps discussed herein, as well as other known equivalents for each such element, feature or step, can be mixed and matched by one of ordinary skill in this art to perform methods in accordance with principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in particular examples.

Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. All references cited herein, including sequences associated with recited GenBank® Accession Numbers available on November 17, 2017, are incorporated by reference in their entirety.

In some examples, the numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain

embodiments are to be understood as being modified in some instances by the term "about" or "approximately." For example, "about" or "approximately" can indicate +/- 20% variation of the value it describes. Accordingly, in some embodiments, the numerical parameters set forth herein are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some examples are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range.

To facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

Administer: To provide or give a subject an agent, such as a copeptin peptide, by any effective route. Administration can be systemic or local. Exemplary routes of administration include, but are not limited to, topical (e.g., transdermal), buccal, vaginal, intranasal, rectal, inhalation, ocular, otic, enteral (e.g. , oral, sublingual, buccal, rectal) and parenteral (e.g., injections (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intratumoral, intraosseous, and intravenous) routes. In some examples, administration is provided directly into a bone or bone marrow. Anemia: A disorder of the blood associated with a decrease in number of red blood cells (RBCs) or less than the normal quantity of hemoglobin in the blood. Anemia can include decreased oxygen-binding ability of each hemoglobin molecule due to deformity or lack in numerical development as in some other types of hemoglobin deficiency. Because hemoglobin (found inside RBCs) normally carries oxygen from the lungs to the capillaries, anemia leads to hypoxia (lack of oxygen) in organs.

The several kinds of anemia are produced by a variety of underlying causes. It can be classified in a variety of ways, for example based on the morphology of RBCs, underlying etiologic mechanisms, and discernible clinical spectra. The three main classes include excessive blood loss (acutely such as a hemorrhage or chronically through low- volume loss), excessive blood cell destruction (hemolysis) or deficient red blood cell production (ineffective hematopoiesis).

Additional types of anemia that can be treated with copeptin using the disclosed methods and compositions include, but are not limited to, iron deficiency anemia, vitamin deficiency anemia (also known as pernicious anemia, e.g., due to deficiencies in folate and/or vitamin B-12, or due to an inability of process vitamin B-12), anemia of chronic disease (e.g., due to cancer, HIV/AIDS, rheumatoid arthritis, kidney disease, Crohn's disease and other chronic inflammatory diseases), aplastic anemia (e.g., due to infection, medication, autoimmune disease, or exposure to toxic chemicals), anemias associated with bone marrow disease (e.g., due to leukemia and

myelofibrosis), hemolytic anemia, sickle cell anemia, thalassemia, malarial anemia, and anemia due to destruction of bone marrow through chemotherapy and/or irradiation (such as those typically used in cancer treatment).

Anemia is typically diagnosed on a complete blood count. Apart from reporting the number of red blood cells and the hemoglobin level, the automatic counters also measure the size of the red blood cells by flow cytometry. Examination of a stained blood smear using a microscope can also be helpful. In some examples, four parameters (RBC count, hemoglobin concentration, MCV and RDW) are measured, allowing others (hematocrit, MCH and MCHC) to be calculated, and compared to values adjusted for age and sex. Some counters estimate hematocrit from direct measurements.

WHO's Hemoglobin thresholds used to define anemia (1 g/dL = 0.6206 mmol/L) are as follows: Age or gender group Hb threshold (g/dl) Hb threshold (mmol/1)

Children (0.5-5.0 yrs) 11.0 6.8

Children (5-12 yrs) 11.5 7.1

Teens (12-15 yrs) 12.0 7.4

Women, non-pregnant (>l5yrs) 12.0 7.4

Women, pregnant 11.0 6.8

Men (>l5yrs) 13.0 8.1

Reticulocyte counts, and the "kinetic" approach to anemia, have become more common. A reticulocyte count is a quantitative measure of the bone marrow's production of new red blood cells.

The reticulocyte production index is a calculation of the ratio between the level of anemia and the extent to which the reticulocyte count has risen in response. If the degree of anemia is significant, even a "normal" reticulocyte count actually may reflect an inadequate response. When the cause is not obvious, clinicians use other tests, such as: ESR, ferritin, serum iron, transferrin, RBC folate level, serum vitamin B12, hemoglobin electrophoresis, renal function tests (e.g. serum creatinine) although the tests can depend on the clinical hypothesis that is being investigated. When the diagnosis remains difficult, a bone marrow examination can be performed.

Arginine Vasopressin (A VP): A nine amino acid long peptide that is released from the brain. The peptide is synthesized as a part of a precursor preprohormone (see FIG. 1A) that is cleaved as it is transported in axons that terminate in the posterior pituitary. The precursor yields AVP, a carrier protein, AVP-neurophysin and the N-terminal glycopeptide copeptin. Once released from nerve endings in the posterior pituitary into the bloodstream, AVP regulates salt and water homeostasis. AVP also acts within the CNS as neuro transmitters.

Hypovolemia or hyperosmolality are strong stimuli for both synthesis and release of AVP from the posterior pituitary. Blood loss resulting in hypovolemia and hypotension is immediately followed by AVP release into the circulation. In dogs, AVP concentrations in plasma rise to a level 40 times greater than normal shortly after the onset of experimental hemorrhagic shock and gradually decline thereafter. In humans, hemorrhage may cause a 50- to a lOO-fold increase in circulating AVP levels paralleled by increases in plasma concentrations of erythropoietin, catecholamines, cortisol, aldosterone, and renin/angiotensin. Arginine Vasopressin (A VP) IB Receptor (AVPR1B): A type of arginine vasopressin receptor involved in the modulation of bone marrow stromal cells, hematopoietic stem cells and progenitor cells. AVPR1B is also known as vasopressin 3 receptor (VPR3) or antidiuretic hormone receptor lb. It is a protein that in humans is encoded by the AVPR1B gene. AVPR1B belongs to the subfamily of G-protein coupled receptors. Its activity is mediated by G proteins, which stimulate a phosphatidylinositol-calcium second messenger system. AVPR1B is a major contributor to homeostasis and the control of water, glucose, and salts in the blood. Arginine vasopressin has four receptors, each of which are located in different tissues and have specific functions. AVPRIB is a G-protein coupled pituitary receptor that has only recently been

characterized because of its rarity.

The 420-amino-acid sequence of the human AVPRIB shares the most overall similarities with the AVP1A, AVP2 and oxytocin receptors. AVPRIB maps to chromosome region lq32 and is a member of the vasopressin/oxytocin family subfamily. Exemplary AVPRIB nucleic acid sequences and amino acid sequences are publically available, see for example, GenBank®

Accession Nos. NM_000707 and NM_0l l924 (Human and Mouse mRNA, respectively) and NP_000698 and NP_036054 (Human and Mouse amino acid sequences, respectively) each of which is incorporated by reference in its entirety as available on February 1, 2018.

Arginine Vasopressin (A VP) RIB Receptor Agonist: A molecule that binds to and activates the vasopressin V1B receptor subtype, modulating hematopoietic stem cell proliferation, for example simulating erythropoiesis. Exemplary AVPRIB agonists include, but are not limited to, d[Cha ⁴ ]AVP; d[Cha ⁴ , Lys ⁸ ]VP, d[Leu4]AVP, and d[Leu ⁴ ,Lys ⁸ ]VP, as disclosed in Manning et al.

(J. Neuroendocrinology 24:609-628, 2012) which is hereby incorporated by reference in its entirety. In some examples, the agonist is a small molecule.

Autoimmune Disease: A condition in which the immune system mistakenly attacks the body. Autoimmune diseases that can be treated with the disclosed methods by administration of copeptin include, but are not limited to: encephalomyelitis (EAE), an inflammatory demyelinating disease of the central nervous system; rheumatoid arthritis, a chronic inflammatory disorder affecting joints; lupus, an inflammatory disease caused when the immune system attacks its own tissues; celiac disease, an immune reaction to gluten; Sjogren's syndrome, an immune system disorder characterized by dry eyes and dry mouth; polymyalgia rheumatic, an inflammatory disorder causing muscle pain and stiffness around the shoulders and hips; multiple sclerosis, a disease in which the immune system erodes the protective covering of nerves; ankylosing spondylitis, an inflammatory arthritis affecting the spine and large joints; type 1 diabetes, a chronic condition in which the pancreas produces little or no insulin; alopecia areata, a sudden hair loss that starts with one or more circular bald patches that may overlap; vasculitis, an inflammation of the blood vessels that causes changes in the blood vessel walls; and temporal arteritis, an inflammation of blood arteries in and around the scalp.

B Cells: B cells, also known as B lymphocytes, are a type of white blood cell of the lymphocyte subtype. They function in the humoral immunity component of the adaptive immune system by secreting antibodies. Additionally, B cells present antigen and secrete cytokines.

Central Diabetes Insipidus: A rare pituitary disease caused by an impairment of neurohypophysial synthesis and/or secretion of arginine vasopressin (A VP). Its clinical syndrome is characterized by polyuria, polydipsia, decreased urine osmolality and increased plasma osmolality.

Chemotherapeutic agent or Chemotherapy: Any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer. In one example, a chemotherapeutic agent is a radioactive compound. In one example, a chemotherapeutic agent is a biologic, such as a monoclonal antibody. In some examples, a subject treated with copeptin using the disclosed methods, is, will be, or was previously treated with chemotherapy. Exemplary chemotherapeutic agents are provided in Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, l4th edition; Perry et ak, Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nd ed., 2000 Churchill Livingstone, Inc; Baltzer and Berkery. (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Lischer Knobf, and Durivage (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Additional specific chemotherapeutic agents are provided herein.

Contacting: Placement in direct physical association; includes both in solid and liquid form. Contacting can occur in vitro with isolated cells (for example in a tissue culture dish or other vessel) or in vivo by administering the agent (such as copeptin) to a subject.

Control: A reference standard. In some examples, a control is a known value or range of values, such as one indicative of a non-anemic or an anemic subject. In some examples, a control is a value or range of values, indicating a response in the absence of a therapeutic agent, such as an amount of hematopoiesis observed without copeptin. Copeptin (CT-proAVP) (e.g., OMIM 192340): A 39-aa peptide derived from a pre-pro- hormone of vasopressin, neurophysin II and copeptin (FIG. 1A). Although A VP is involved in multiple cardiovascular and renal pathways and functions, measurements are not commonly used in clinical practice. On the other hand, copeptin can be immunologically tested with ease, and therefore is used as a vasopressin surrogate.

Copeptin sequences are publically available, for example from the GenBank® sequence database (e.g., Accession Nos. NP_00048l.2, NP_033862.l, NP_789824.l, and NP_058688.2 provide exemplary pre-pro- vasopressin precursor protein sequences, which include a C-terminal 39- aa copeptin sequence). One of ordinary skill in the art can identify additional copeptin nucleic acid and protein sequences, including copeptin variants.

Specific examples of copeptin sequences are provided in SEQ ID NOS: 1-4 (also see FIG. 1B). Variant copeptin sequences, such as one having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any of SEQ ID NOS: 1-4), retain the ability to modulate hematopoiesis (e.g., stimulate/increase hematopoiesis in stress conditions, such as depleted RBCs, or decrease hematopoiesis in non-stress/normal/steady-state conditions,), such as erythropoiesis and/or lymphopoiesis.

Copeptin is disclosed herein to modulate hematopoiesis (such as increase or decrease), such as erythropoiesis, lymphopoiesis, and/or platelet formation; modulate the immune response, such as T cell and B cell formation, and stimulate bone formation, such as osteoblast, osteocyte, chondrocyte, adipocyte formation, or combinations thereof.

Differentiation: The process whereby relatively unspecialized cells (such as embryonic stem cells, HSCs, or other stem cells) acquire specialized structural and/or functional features characteristic of mature cells. Similarly,“differentiate” refers to this process. Typically, during differentiation, cellular structure alters and tissue-specific proteins appear.

Effective amount or Therapeutically effective amount: The amount of agent (such as copeptin, alone or with one or more other therapeutic agents) sufficient to induce a desired response, such as to prevent, treat, reduce and/or ameliorate the symptoms and/or underlying causes of any of a disorder or disease, or to increase the number of cells, such as to increase the proliferation of cells, including stem cells, such as HSCs. In one example, an“effective amount” is sufficient to reduce or eliminate a symptom of a disease, such as a sign or symptom of anemia, polycythemia, an autoimmune disease, or infection. In another example, an effective amount is an amount sufficient to overcome the disease itself. In a further example, an effective amount of copeptin is an amount that produces a statistcally significant increase in the number of HSCs (such as erythroblastss) in culture or in an in vivo model of anemia (or other condition where RBCs are decreased) as compared to a control, such as a culture or subject not treated with copeptin or treated with vehicle alone. In a further example, an effective amount of copeptin is an amount that produces a statistcally significant decrease in the number of HSCs (such as erythroblastss) in culture or in an in vivo model of a normal subject (e.g., condition where RBCs are at normal levels)as compared to a control, such as a culture or subject not treated with copeptin or treated with vehicle alone.

The condition or disease, such as anemia, polycythemia, an autoimmune disease, or infection, does not need to be completely inhibited for the pharmaceutical preparation to be effective. Treatment can include slowing the progression of the disease temporarily, but can also include halting or reversing the progression of the disease permanently. For example, a pharmaceutical preparation can alleviate one or more signs or symptoms associated with anemia by increasing HSC proliferation (such as hematopoiesis, e.g., erythropoiesis and/or lymphopoiesis, for example in the spleen or bone marrow) by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, even at least 100%, as compared to proliferation in the absence of the pharmaceutical preparation.

Effective amounts of copeptin, alone or with one or more other therapeutic agents, can be determined in many different ways, such as assaying for a reduction in of one or more signs or symptoms associated with the condition, such as anemia or hemorrhage, in the subject or measuring the level of one or more molecules associated with the condition to be treated. Effective amounts also can be determined through various in vitro, in vivo or in situ assays, including the assays described herein.

Erythrocyte: The most common type of blood cell and the vertebrate organism's principal means of delivering oxygen (0 ₂ ) to the tissues via the blood flow through the circulatory system.

An erythrocyte is also known as a red blood cell (RBC).

Erythropoiesis: The process by which red blood cells (erythrocytes) are produced. It can be stimulated by decreased O2 in circulation, which is detected by the kidneys, which then secrete erythropoietin (EPO). EPO stimulates proliferation and differentiation of red cell precursors (such as multipotent hematopoietic stem cells, normoblasts, and reticulocytes), which activates increased erythropoiesis in the hemopoietic tissues, ultimately producing red blood cells. In postnatal birds and mammals (including humans), this usually occurs within the red bone marrow. In the early fetus, erythropoiesis takes place in the mesodermal cells of the yolk sac. By the third or fourth month, erythropoiesis moves to the spleen and liver. After seven months, erythropoiesis occurs in the bone marrow. Increased level of physical activity can cause an increase in erythropoiesis. However, in humans with certain diseases and in some animals, erythropoiesis also occurs outside the bone marrow, within the spleen or liver. This is termed extramedullary erythropoiesis.

Erythropoietins available for use as therapeutic agents (for example in combination with copeptin) include Epogen/Procrit (epoetin alfa) and Aranesp (darbepoetin alfa). Recombinant EPO has a variety of glycosylation patterns giving rise to alfa, beta, delta, and omega forms. In some examples, one or more of these are used in combination with copeptin in the methods provided herein, for example to increase erythropoiesis in a subject.

epoetin alfa: Darbepoetin (Aranesp); Epocept (Lupin pharma); Nanokine (Nanogen Pharmaceutical biotechnology); Epofit (Intas pharma); Epogen (Amgen); Epogin; Eprex (Janssen- Cilag); Binocrit (Sandoz; Procrit).

epoetin beta: NeoRecormon (Hoffmann-La Roche); Recormon; Methoxy polyethylene glycol-epoetin beta (Mircera) by Roche

epoetin delta: Dynepo® erythropoiesis stimulating protein (Shire plc)

epoetin omega: Epomax

epoetin zeta (biosimilar forms for epoetin alpha): Silapo (Stada); Retacrit (Hospira)

Miscellaneous: EPOTrust (Panacea Biotec Ltd); Erypro Safe (Biocon Ltd.); Repoitin (Serum Institute of India Limited); Vintor (Emcure Pharmaceuticals); Erykine, (Intas

Biopharmaceutica); Wepox (Wockhardt Biotech); Espogen (LG life sciences); ReliPoietin

(Reliance Life Sciences); Shanpoietin (Shantha Biotechnics Ltd); Zyrop (Cadila Healthcare Ltd.); EPIAO (rHuEPO) (Shenyang Sunshine Pharmaceutical Co.. LTD. China); Darbepoetin alfa is a form created by five substitutions (Asn-57, Thr-59, Val-l l4, Asn-ll5 and Thr-l l7) that create two new N-glycosylation sites; and Novel erythropoiesis-stimulating protein (NESP; Macdougall IC, Semin. Nephrol. 20 (4): 375-81, 2000).

Fc-Fusions: Homodimers in which a fragment crystallizable (Fc) domain of an antibody is covalently linked to another protein (such as copeptin). In some examples, the fusion partner is directly attached to the flexible hinge, the length and sequence of which varies between different IgG subclasses. Hemorrhage: The loss of blood or blood escaping from the circulatory system.

Hemorrhaging (bleeding) can arise due to either traumatic injury, underlying medical condition, or a combination thereof.

Immune Response: The reaction to and interaction with substances interpreted by the body as not-self. The immune response depends on a functioning thymus and the conversion of stem cells to B and T lymphocytes. These lymphocytes contribute to antibody production, cellular immunity, and immunologic memory. Pathologic conditions associated with an abnormal immune response (immunopathy) may result from immunodepression, excessive production of gamma globulins, overreaction to antigens of extrinsic origin, or abnormal response of the body to its own cells and tissues. Factors that may cause or contribute to suppression of the immune response include (1) congenital absence of the thymus or of the stem cells that are precursors of B and T lymphocytes; (2) malnutrition, in which there is a deficiency of the specific nutrients essential to the life of antibody-synthesizing cells; (3) cancer, viral infections, and extensive burns, all of which overburden the immune response mechanisms and rapidly deplete the supply of antigen-specific antibody; (4) certain drugs, including alcohol and heroin, some antibiotics, antipsychotics, and the antineoplastics used in the treatment of cancer. Overproduction of gamma globulins is manifested by an excessive proliferation of plasma cells (multiple myeloma). Hypersensitivity is the result of an overreaction to substances entering the body.

Inhibiting a disease or condition: Reducing, slowing, or even stopping the development of a disease or condition, for example, in a subject who is at risk for a disease or who has a particular disease, such as anemia.

Lymphopoiesis: The generation of lymphocytes, a type of white blood cell (WBC). T-cells are formed in bone marrow then migrate to the cortex of the thymus to undergo maturation in an antigen-free environment. B-cells are formed and mature in the bone marrow and spleen.

Pathogenesis in lymphopoiesis can result in a lymphoproliferative disorder, such as a lymphoma or lymphoid leukemia.

Osteoporosis: A disease in which the density and quality of bone are reduced. As bones become more porous and fragile, the risk of fracture is greatly increased. The loss of bone occurs silently and progressively. Often there are no symptoms until the first fracture occurs.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers useful in this disclosure are conventional. Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, l9th Edition (1995), describes compositions and formulations suitable for pharmaceutical delivery of the compositions herein disclosed (such as copeptin). For example copeptin or other therapeutic agents can be administered in the presence of one or more

pharmaceutically acceptable carriers, including a non-natural or natural pharmaceutically acceptable carrier molecule.

The nature of the carrier can depend on the particular mode of administration being employed. For instance, parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for instance, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Embodiments of other pharmaceutical compositions can be prepared with conventional

pharmaceutically acceptable carriers, adjuvants, and counter-ions, as would be known to those of skill in the art. The compositions in some embodiments are in the form of a unit dose in solid, semi-solid, and liquid dosage forms, such as tablets, pills, capsules, lozenges, powders, liquid solutions, or suspensions.

Polycythemia: A disease state in which the proportion of blood volume that is occupied by red blood cells increases, for example is greater than 55% of the blood volume. Blood volume proportions can be measured as hematocrit level. It can be due to an increase in the number of red blood cells ("absolute polycythemia") or to a decrease in the volume of plasma ("relative polycythemia"). In some examples, copeptin or an antagonist of copeptin is used to treat polycythemia using the methods provided herein.

Sequence identity of amino acid sequences: The similarity between amino acid (or nucleotide) sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 13:231, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol.

215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.

Homologs and variants of a copeptin or AVP protein disclosed herein are typically characterized by possession of at least about 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity counted over the full length alignment with the amino acid sequence using the NCBI Blast 2.0, gapped blastp set to default parameters. For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or at least 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.

Thus, a copeptin protein used with the disclosed methods can have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, 2, 3, or 4, and retain the ability to

stimulate/increase hematopoiesis, such as erythropoiesis and/or lymphopoiesis. Stem cell: A cell that can generate a fully differentiated functional cell of a more than one given cell type. The role of stem cells in vivo is to replace cells that are destroyed during the normal life of an animal. Generally, stem cells can divide without limit and are totipotent or pluripotent. After division, the stem cell may remain as a stem cell, become a precursor cell, or proceed to terminal differentiation. A nervous system (NS) stem cell is, for example, a cell of the central nervous system that can self-renew and can generate astrocytes, neurons and

oligodendrocytes .

A hematopoietic stem cell (HSC) is, for example, a cell that gives rise to all other blood cells. They give rise to the myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoid lineages (T-cells, B-cells, NK-cells). The hematopoietic tissue contains cells with long-term and short-term regeneration capacities and committed multipotent, oligopotent, and unipotent progenitors. HSCs constitute 1:10.000 of cells in myeloid tissue. HSCs are a heterogeneous population. Three classes of stem cells exist, distinguished by their ratio of lymphoid to myeloid progeny (L/M) in blood. Myeloid-biased (My-bi) HSC have low L/M ratio (>0, <3), whereas lymphoid-biased (Ly-bi) HSC show a large ratio (>10). The third category consists of the balanced (Bala) HSC for which 3 < L/M < 10. Only the myeloid-biased and -balanced HSCs have durable self-renewal properties. In addition, serial transplantation studies have shown that each subtype preferentially re-creates its blood cell type distribution, suggesting an inherited epigenetic program for each subtype. HSCs can be identified and characterized based upon morphology, presence and/or absence of particular markers and functional assays, such as the cobblestone area-forming cell (CAFC) assay.

A bone marrow stem cell is, for example, an adult, mesoderm-derived cell that is capable of generating cells of mesenchymal lineages, typically of two or more mesenchymal lineages, e.g., osteocytic (bone), chondrocytic (cartilage), myocytic (muscle), tendonocytic (tendon), fibroblastic (connective tissue), adipocytic (fat) and stromogenic (marrow stroma) lineage. Bone marrow stem cells are present in or (partly) isolated from a sample of bone marrow. A sample of bone marrow may be obtained, e.g., from iliac crest, femora, tibiae, spine, rib or other medullar spaces of a subject. Bone marrow stem cells encompass any and all subtypes thereof, such as without limitation, "rapidly self-renewing cells" RS-l or RS-2 as described in Colter et al. ( PNAS 97(7): 3213-8, 2000); "side population" (SP) cells as described by Goodell et al. (Nat Med 3(12): 1337-45, 1997); osteogenic precursor (OP) cells which are initially identified by their low density (e.g., upon density gradient centrifugation), non-adherent nature and low-level of expression of osteogenic markers (as described by Long et al.1995. J Clin Invest. 95(2): 881-7; U.S. Pat. No. 5,972,703); primitive precursor cells which can generate cells of both the haematopoietic and non- haematopoietic lineages as described by Krause et al. ( Cell 105: 369-377, 2001) and Dominici et al. (PNAS 101(32): 11761-6, 2004); and others. Bone cells include osteoblasts (bone forming cells), osteoclasts (bone removing cells), and osteocytes (bone maintaining cells). Cartilage is made up of chondrocytes. Adipocytes are fat cells.

Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals, as well as birds (such as chickens and turkeys), fish, and reptiles. Exemplary subjects include mammals, such as human and non-human primates, rats, mice, dogs, cats, rabbits, cows, pigs, goats, horses, and the like.

T Cells: T cells originate in the bone marrow and mature in the thymus, where they multiply and differentiate into helper, regulatory, or cytotoxic T cells or become memory T cells. These T cells are then sent to peripheral tissues or circulate in the blood or lymphatic system. Once stimulated by the appropriate antigen, helper T cells secrete cytokines, which stimulate the differentiation of B cells into plasma cells (antibody-producing cells). Regulatory T cells act to control immune reactions. Cytotoxic T cells, which are activated by various cytokines, bind to and kill infected cells and cancer cells.

Under conditions sufficient to: A phrase that is used to describe any environment that permits the desired activity.

Methods of Modulating Hematopoiesis

Methods of modulating, for example by stimulating or inhibiting, hematopoiesis, are disclosed. Such methods can include modulating (such as increasing or decreasing) erythropoiesis, lymphopoiesis, or both. Thus, the disclosed methods modulate (such as increase or decrease) hematopoietic (blood) stem cell (such as a bone marrow stromal stem cells) proliferation and/or differentiation, such as modulate (such as increase or decrease) proliferation and/or differentiation of cells that gives rise to myeloid cells (e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, and dendritic cells), and lymphoid cells (e.g., T-cells, B-cells, and NK-cells). In some examples, copeptin is used to regulate cell production within the bone marrow and regulate the number of circulating blood cells.

In some examples, such methods are used to treat or prevent a disorder wherein increased hematopoiesis is desired, such as increased erythropoiesis, increased lymphopoiesis, or both, for example anemia/reduced RBC (e.g., due to bleeding or chemotherapy or radiation therapy), or reduced WBC (e.g., due to infection, chemotherapy, or radiation therapy). In some examples, the disclosed methods lessen, ameliorate, eliminate, prevent, or inhibit hematopoiesis, such as erythropoiesis, or treat or prevent a disorder or condition associated with a pathological increase in RBC production, such as polycythemia.

Also disclosed herein are methods of modulating (such as increasing or decreasing) an immune response in a subject. Such methods can include administering to the subject a therapeutically effective amount of copeptin. Such methods can be used to treat or prevent a disorder wherein modulation (such as decreasing) of the immune system of a subject is desired, such as treatment of an autoimmune disease. In some examples, the disclosed methods regulate T cell formation, B cell formation, or both, to treat or prevent an autoimmune disease, such as encephalomyelitis (EAE), lupus, rheumatoid arthritis, celiac disease, Sjogren's syndrome, polymyalgia rheumatic, multiple sclerosis, ankylosing spondylitis, alopecia areata, vasculitis or temporal arteritis. These methods may further include administering to the subject a therapeutically effective amount of one or more additional therapeutic agents, such as AVP, AVPR1B agonist(s) (such as d[Cha ⁴ ]AVP; d[Cha ⁴ , Lys ⁸ ]VP, d[Leu4]AVP, or d[Leu ⁴ ,Lys ⁸ ]VP), EPO, or any combination thereof. In some examples, the method includes administering a therapeutically effective amount of EPO, AVPR1B agonist(s) (such as d[Cha ⁴ ]AVP; d[Cha ⁴ , Lys ⁸ ]VP, d[Leu4]AVP, or d[Leu ⁴ ,Lys ⁸ ]VP), and copeptin.

Also disclosed herein are methods of treating diabetes in a subject, which includes administering to the subject a therapeutically effective amount of copeptin. In some examples, the method further includes administering to the subject a therapeutically effective amount of one or more other therapeutic agents, such as therapeutically effective amount of AVP, an AVPR1B agonist (such as, but not limited to, d[Cha ⁴ ]AVP; d[Cha ⁴ , Lys ⁸ ]VP, d[Leu4]AVP, or

d[Leu ⁴ ,Lys ⁸ ]VP), EPO, or any combination thereof. In some examples, the method includes administering a therapeutically effective amount of EPO, AVPR1B agonist(s), and copeptin. Such methods can be used to treat or prevent central diabetes insipidus.

Also disclosed herein are methods of stimulating bone, cartilage or both in a subject, which include administering to the subject a therapeutically effective amount of copeptin. In some examples, the method further includes administering to the subject a one or more other therapeutic agents, such as a therapeutically effective amount of AVP, an AVPR1B agonist (such as, but not limited to, d[Cha ⁴ ]AVP; d[Cha ⁴ , Lys ⁸ ]VP, d[Leu4]AVP, or d[Leu ⁴ ,Lys ⁸ ]VP), EPO, or any combination thereof. In some examples, the method includes administering a therapeutically effective amount of EPO, AVPR1B agonist(s), and copeptin. Such methods can be used to treat or prevent a fracture, or a disease or condition associated with insufficient bone mass density, such as osteopenia and osteoporosis. In some examples, the disclosed methods stimulate, improve, ameliorate or increase bone mass density or cartilage formation. These methods may further include administering to the subject a therapeutically effective amount of one or more additional therapeutic agents, such as A VP, AVPR1B, EPO, or any combination thereof.

In some examples, such methods increase or decrease cell proliferation (such as

proliferation of HSCs, bone marrow stromal stem cells, RBCs, WBCs, or combinations thereof) by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, such as an increase of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 200% or more), or decrease cell proliferation (such as proliferation of HSCs, bone marrow stromal stem cells, RBCs, WBCs, or combinations thereof), such as a decrease of at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, such as a decrease of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100%, for example relative to a control (e.g., no treatment with an effective amount of copeptin).

In some examples, such methods increase hematopoiesis by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, such as an increase of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 200% or more), or decrease hematopoiesis such as a decrease of at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, such as a decrease of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,

98%, or 100%, for example relative to a control (e.g., no treatment with an effective amount of copeptin). In some examples, such methods increase erythropoiesis by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, such as an increase of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 200% or more), or decrease erythropoiesis by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, such as an decrease of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%), for example relative to a control (e.g., no treatment with an effective amount of copeptin).

In some examples, such methods increase a number of orthochromatic erythroblasts in a subject (for example in the bone marrow or spleen) by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, such as an increase of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 200% or more), or decrease a number of orthochromatic erythroblasts (for example in the bone marrow) in a subject by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, such as a decrease of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%), for example relative to a control (e.g., no treatment with an effective amount of copeptin).

In some examples, such methods increase a number of polychromatic erythroblasts in a subject (for example in the bone marrow or spleen) by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, such as an increase of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 200% or more), or decrease a number of polychromatic erythroblasts (for example in the bone marrow) in a subject by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, such as a decrease of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%), for example relative to a control (e.g., no treatment with an effective amount of copeptin).

In some examples, such methods increase a number of erythroid precursor cells in a subject (for example in the bone marrow or spleen) by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, such as an increase of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 200% or more), or decrease a number of erythroid precursor cells (for example in the bone marrow) in a subject by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, such as a decrease of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%), for example relative to a control (e.g., no treatment with an effective amount of copeptin).

In some examples, such methods increase a number of RBCs in a subject by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, such as an increase of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 200% or more), or decrease a number of RBCs in a subject by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, such as a decrease of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%), for example relative to a control (e.g., no treatment with an effective amount of copeptin).

In some examples, such methods increase a number of reticulocytes in a subject (for example in the circulating blood) by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, such as an increase of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 200% or more), or decrease a number of reticulocytes in a subject by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, such as a decrease of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%), for example relative to a control (e.g., no treatment with an effective amount of copeptin).

In some examples, such methods increase lymphopoiesis by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, such as an increase of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 200% or more), or decrease lymphopoiesis by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, such as a decrease of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g. , a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%), for example relative to a control (e.g. , no treatment with an effective amount of copeptin).

In some examples, such methods increase a number of WBCs in a subject by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, such as an increase of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 200% or more), or decrease a number of WBCs in a subject by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, such as a decrease of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%), for example relative to a control (e.g., no treatment with an effective amount of copeptin).

In some examples, such methods increase a number of T-cells (such as T-helper cells, T- cytotoxic cells, T-memory cells, T-suppressor cells, T-regulatory cells, natural killer T-cells, or combinations thereof) in a subject by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, such as an increase of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 200% or more), or decrease a number of T-cells (such as T-helper cells, T-cytotoxic cells, T-memory cells, T-suppressor cells, T-regulatory cells, natural killer T-cells, or combinations thereof) in a subject by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, such as a decrease of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%), for example relative to a control (e.g., no treatment with an effective amount of copeptin).

In some examples, such methods increase a number of B-cells in a subject by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, such as an increase of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 200% or more), or decrease a number of B-cells in a subject by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, such as a decrease of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%), for example relative to a control (e.g., no treatment with an effective amount of copeptin).

In some examples, such methods increase a number of natural killer cells in a subject by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, such as an increase of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,

95%, 100%, 200% or more), , or decrease a number of natural killer-cells in a subject by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, such as a decrease of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%), for example relative to a control (e.g., no treatment with an effective amount of copeptin). In some examples, such methods increase a number of dendritic cells in a subject by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, such as an increase of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g. , a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 200% or more), , or decrease a number of dendritic cells in a subject by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, such as a decrease of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%), for example relative to a control (e.g., no treatment with an effective amount of copeptin).

In some examples, such methods increase a number of myeloid cells in a subject by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, such as an increase of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g. , a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,

95%, 100%, 200% or more), or decrease a number of myeloid cells a subject by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, such as a decrease of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g. , a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%), for example relative to a control (e.g., no treatment with an effective amount of copeptin). In some examples, combinations of these effects are achieved.

In some examples, such methods increase a number of osteoblasts in a subject by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, such as an increase of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,

95%, 100%, 200% or more), for example relative to a control (e.g., no treatment with an effective amount of copeptin). In some examples, such methods increase a number of osteocytes in a subject by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, such as an increase of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g. , a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 200% or more), for example relative to a control (e.g., no treatment with an effective amount of copeptin).

In some examples, such methods increase a number of chondrocytes in a subject by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, such as an increase of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g. , a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 200% or more), for example relative to a control (e.g., no treatment with an effective amount of copeptin).

In some examples, such methods increase a number of adipocytes in a subject by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, such as an increase of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,

95%, 100%, 200% or more), for example relative to a control (e.g., no treatment with an effective amount of copeptin).

In some examples, such methods decrease a number of osteoclasts in a subject by at least about 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, such as an decrease of 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%), for example relative to a control (e.g., no treatment with an effective amount of copeptin). In some examples, combinations of these effects are achieved.

Copeptin

The precursor molecule of vasopressin includes three proteins: AVP, AVP-neurophysin and a C-terminal glycopeptide called copeptin (see FIG. 1A). Copeptin is a 39 amino acid glycosylated hormone that has been studied for almost 50 years, but no function or receptor has been identified until now.

In some examples, the copeptin used in the disclosed methods is glycosylated. In some examples, the copeptin used in the disclosed methods is not glycosylated.

In some examples, the copeptin peptide used in the disclosed methods consists of the sequence shown in SEQ ID NO: 1, 2, 3 or 4. However, variant copeptin proteins, including variants of the sequences shown in SEQ ID NO: 1, 2, 3 or 4, can be used, such as one having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOS: 1, 2, 3 and 4. For example, by aligning the sequences of SEQ ID NO: 1, 2, 3 and 4 (see FIG. 1B), one can identify places where amino acid substitutions are likely to be tolerated (e.g., where the aa are less conserved between species), and where amino acid substitutions are not likely to be tolerated (e.g., where the aa conserved between species). For example changes at amino acids 1, 5, 6, 7, 8, 10, 11, 12, 13, 17- 26, 29, 34, 36, and 39 of SEQ ID NOS: 1-4 are not likely to be tolerated, while changes at amino acids 2, 4, 9, 15-16, 28, 30, 31, 33, and 38 of SEQ ID NOS: 1-4 are more likely to be tolerated. Positions 3, 14, 27, 32, 35 and 38 of SEQ ID NOS: 1-4 may tolerate conservative substitutions.

Copeptin variants can contain one or more mutations, such as a single insertion, a single deletion, a single substitution, or combinations thereof. Thus, if a variant copeptin includes one or more amino acid deletions and/or insertions, the variant copeptin may not be 39 amino acids, but is useful in the disclosed methods if it can modulate (e.g., stimulate or increase) hematopoiesis. In some examples, the variant copeptin protein includes 1-5 insertions, 1-5 deletions, 1-5

substitutions, or any combination thereof (e.g., single insertion together with 1-4 substitutions). In some examples, the method uses a variant copeptin having 1, 2, 3, 4, or 5 amino acid changes. In some examples, SEQ ID NO: 1, 2, 3 or 4, includes 1-5 insertions, 1-5 deletions, 1-5 substitutions, or any combination thereof. In some examples, the disclosure provides a variant of any of SEQ ID NOS: 1, 2, 3 or 4, having 1, 2, 3, 4, or 5 amino acid changes.

One type of modification or mutation includes the substitution of amino acids for amino acid residues having a similar biochemical property, that is, a conservative substitution (such as 1-5, 1-4, 1-3, or 1-2 conservative substitutions). Typically, conservative substitutions have little to no impact on the activity of a resulting peptide. For example, a conservative substitution is an amino acid substitution in SEQ ID NO: 1, 2, 3 or 4, does not substantially affect the ability of the copeptin to stimulate/increase hematopoiesis, such as erythropoiesis and/or lymphopoiesis. An alanine scan can be used to identify which amino acid residues in a variant copeptin protein, such as SEQ ID NO: 1, 2, 3 or 4, can tolerate an amino acid substitution. In one example, hematopoiesis activity of copeptin, or any of SEQ ID NO: 1, 2, 3 or 4, is not altered by more than 25%, for example not more than 20%, for example not more than 10%, when an alanine, or other conservative amino acid, is substituted for 1-5 native amino acids. Examples of amino acids which may be substituted for an original amino acid in copeptin and which are regarded as conservative substitutions include: Ser for Ala; Lys for Arg; Gln or His for Asn; Glu for Asp; Ser for Cys; Asn for Gln; Asp for Glu; Pro for Gly; Asn or Gln for His; Leu or Val for Ile; Ile or Val for Leu; Arg or Gln for Lys; Leu or Ile for Met; Met, Leu or Tyr for Phe; Thr for Ser; Ser for Thr; Tyr for Trp; Trp or Phe for Tyr; and Ile or Leu for Val.

More substantial changes can be made by using substitutions that are less conservative, e.g., selecting residues that differ more significantly in their effect on maintaining: (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation; (b) the charge or hydrophobicity of the polypeptide at the target site; or (c) the bulk of the side chain. The substitutions that in general are expected to produce the greatest changes in polypeptide function are those in which: (a) a hydrophilic residue, e.g., serine or threonine, is substituted for (or by) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, valine or alanine; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysine, arginine, or histidine, is substituted for (or by) an electronegative residue, e.g., glutamic acid or aspartic acid; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine. The effects of these amino acid substitutions (or other deletions or additions) can be assessed by analyzing the function of the variant copeptin, such as any of SEQ ID NOS: 1, 2, 3 or 4, by analyzing the ability of the variant copeptin to stimulate/increase hematopoiesis, such as erythropoiesis and/or lymphopoiesis.

Tiie copeptin used in the disclosed methods, compositions, and kits, can he natural copeptin or synthesized or recombinant copeptin. The recombinant copeptin can include modifications of the native copeptin sequence, such as amino acid substitutions, deletions or insertions, glycosylation and/or covalent linkage to unrelated proteins (e.g., a protein tag), as long as the recombinant copeptin retains its biological properties. These variations in sequence can be naturally occurring variations or they can be engineered through the use of genetic engineering techniques. Examples of such techniques are found in see, e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4 ^th ed., Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013, both of which are incorporated herein by reference in their entirety.

The recombinant copeptin disclosed herein can include additional amino acid substitutions that are known to increase the stability or expression of the copeptin.

In some embodiments, the recombinant copeptin can include one or more amino acid substitutions compared to a corresponding native copeptin sequence. For example, in some embodiments, the recombinant copeptin includes up to 10 (such as up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions (such as conservative amino acid substitutions) compared to a native copeptin sequence, such the sequence of a copeptin set forth as SEQ ID NO: 1. The simplest modifications involve the substitution of one or more amino acids for amino acids having similar biochemical properties, such as conservative amino acid substitutions. Such substitutions are likely to have minimal impact on the activity of the resultant copeptin. In some embodiments, the recombinant copeptin can be joined at either end to other unrelated sequences (for example non-copeptin protein sequences, non- viral envelope, or non- viral protein sequences). The recombinant copeptin can be derivatized or linked to another molecule (such as another peptide or protein).

In several embodiments, the copeptin or fragment thereof used is soluble in aqueous solution. In some embodiments, the copeptin dissolves to a concentration of at least 0.5 mg/ml (such as at least 1.0 mg/ml, 1.5 mg/ml, 2.0 mg/ml, 3.0 mg/ml, 4.0 mg/ml or at least 5.0 mg/ml) in aqueous solution (such as phosphate buffered saline (pH 7.4) or 350 mM NaCl (pH 7.0)) at room temperature (e.g., 20-22°C) and remains dissolved for at least 12 hours (such as at least 24 hours, at least 48 hours, at least one week, at least two weeks, at least one month, or more time). In one embodiment, the phosphate buffered saline includes NaCl (137 mM), KC1 (2.7 mM), Na ₂ HP0 ₄ (10 mM), KΉ2RO4 (1.8 mM) at pH 7.4. In some embodiments, the phosphate buffered saline further includes CaCh (1 mM) and MgCh (0.5 mM). The concentration of the protein dissolved in an aqueous solution can be tested over time using standard methods.

In certain embodiments, the copeptin or fragment thereof used may be further modified to contain additional non-proteinaceous moieties, such as water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-l,3-dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n- vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to copeptin may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the copeptin to be improved or altered, whether the copeptin derivative will be used in a therapy under defined conditions, etc.

In some embodiments, the copeptin or fragment thereof can be membrane anchored. In such embodiments, the copeptin is linked to a transmembrane domain, and/or a transmembrane domain and cytosolic tail. In some embodiments, one or more peptide linkers (such as a gly-ser linker, for example, a 10 amino acid glycine- serine peptide linker) can be used to link copeptin to the transmembrane domain.

Additional aspects of the disclosure include analogs, derivatives, and mimetics based on the amino acid sequence of the disclosed therapeutic copeptin. Typically, mimetic compounds are synthetic compounds having a three-dimensional structure (of at least part of the mimetic compound) that mimics, for example, the primary, secondary, and/or tertiary structural, and/or electrochemical characteristics of a selected peptide, structural domain, active site, or binding region (e.g., a homotypic or heterotypic binding site, a catalytic active site or domain, a receptor or ligand binding interface or domain, or a structural motif) thereof. The mimetic compound will often share a desired biological activity with a native peptide, as discussed herein. Typically, at least one subject biological activity of the mimetic compound is not substantially reduced in comparison to, and is often the same as or greater than, the activity of the native peptide on which the mimetic was modeled.

A variety of techniques are available for constructing peptide mimetics with the same, similar, increased, or reduced biological activity as the corresponding native peptide. Often these analogs, variants, derivatives and mimetics will exhibit one or more desired activities that are distinct or improved from the corresponding native peptide, for example, improved characteristics related to the modulation of cell growth.

The copeptin sequences disclosed herein can be derivatized or linked to another molecule (such as another peptide or protein). In general, the polypeptide or portion thereof is derivatized such that the biological activity of the therapeutic copeptin is not affected adversely by the derivatization or labeling. For example, the polypeptide can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as an antibody (for example, to form a bispecific polypeptide), a detection agent, a pharmaceutical agent, and/or a protein or peptide that can mediate associate of the polypeptide with another molecule (such as a streptavidin core region or a polyhistidine tag).

In additional embodiments, the copeptin is linked to an N-terminal or C-terminal signal peptide sequence. Expression of such a polypeptide in a cell results in entry of the copeptin into the secretory system of the cell. Exemplar signal peptide sequences are provided in Blobel and Dobberstein, J. Cell Bio., 67:835-51, 1975; U.S. Pat. Nos. 5,514, 590; 5,580,758; 5,726,038;

5,932,445; and 7,527,947. In some embodiments, the signal sequence is derived from a secreted protein. In some embodiments, the signal peptide sequence is the signal peptide of MANF (MWATQGLAVALALSVLPGSRA; SEQ ID NO: 5 or CDNF

(MWCASPVAVVAFCAGLLVSHP; SEQ ID NO: 6), the signal peptide of BIP

(MKLSLVAAMLLLLSAARA (SEQ ID NO: 7; residues 1-18 of GENBANK® Accession No NP_005338.l); the signal peptide of gaussia luciferase (MGVKVLFALICIAVAEA; SEQ ID NO: 8); the signal peptide of albumin (MKWVTFISLLFLFSS AY S ; SEQ ID NO: 9; see GENBANK® Accession Nos. NP_000468.l, and NM_000477.5); the signal peptide of GDNF

(MKLWD VV A V CLVLLHT AS A ; SEQ ID NO: 10, see GENBANK® Accession Nos.

NP_000505.l and NM_0005l4.3); or the signal peptide of BDNF (MTILFLTMVISYFGCMKA, SEQ ID NO: 11; see GENBANK® Accession Nos. NM_l70735.5 and NP_73393l.l).

Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH ₂ ) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on a copeptin to result in the binding of the effector molecule.

Alternatively, the polypeptide is derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford, IL. The linker can be any molecule used to join the copeptin to the effector molecule. The linker is capable of forming covalent bonds to both the polypeptide and to the effector molecule. Suitable linkers include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where effector molecule is a polypeptide, the linkers may be joined to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.

Effector molecules, such as therapeutic, diagnostic, or detection moieties can be linked to copeptin. Both covalent and noncovalent attachment means may be used. The procedure for attaching an effector molecule to a polypeptide varies according to the chemical structure of the effector. In some circumstances, it is desirable to free the effector molecule from copeptin when the conjugate has reached its target site. Therefore, in these circumstances, conjugates will include linkages that are cleavable in the vicinity of the target site. Cleavage of the linker to release the effector molecule from copeptin may be prompted by enzymatic activity or conditions to which the conjugate is subjected either inside the target cell or in the vicinity of the target site. In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules) drugs, toxins, and other agents, one skilled in the art will be able to determine a suitable method for attaching a given agent to copeptin.

Copeptin can be labeled with a detectable moiety. Useful detection agents include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5- dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like. Bioluminescent and fluorescent markers are also of use, such as luciferases, green fluorescent protein (GFP), yellow fluorescent protein (YFP) and red fluorescent protein (RFP). Copeptin can also be labeled with enzymes that are useful for detection, such as horseradish peroxidase, b- galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. A detectable enzyme can be detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is visually detectable. Copeptin may also be labeled with biotin, and detected through indirect measurement of avidin or streptavidin binding. It should be noted that the avidin itself can be labeled with an enzyme or a fluorescent label.

Copeptin may be labeled with a magnetic agent, such as gadolinium. Copeptin can also be labeled with lanthanides (such as europium and dysprosium), and manganese. Paramagnetic particles such as superparamagnetic iron oxide are also of use as labels. Copeptin may also be labeled with a predetermined polypeptide epitopes recognized by a secondary reporter (such as leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

Copeptin can also be labeled with a radiolabeled amino acid. The radiolabel may be used for both diagnostic and therapeutic purposes. For instance, the radiolabel may be used to detect copeptin by x-ray, emission spectra, or other diagnostic techniques. Examples of labels include, but are not limited to: ¾ ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, "Tc, ^m In, ¹²⁵ I, ¹³¹ I.

Copeptin can also be derivatized with a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These groups may be useful to improve the biological characteristics of copeptin, such as to increase serum half-life or to increase tissue binding.

In some examples, the copeptin used in the disclosed methods and compositions is linked to one or more immunoglobulin Fc domains, such as the Fc of human IgGl (for example those disclosed in Czajkowsky et ah, EMBO Mol Med 4:1015-28, 2012, herein incorporated by reference in its entirety).

Subjects

Exemplary subjects that can be treated with the disclosed methods include vertebrates, such as mammals, including primates. In some examples, the subject is a human. However, other animals can be treated, including cats, dogs, livestock, reptiles, fish, birds, and the like.

In some examples, the subject has anemia, or is at risk to develop anemia. Thus, the methods can be used to treat or prevent anemia, such as anemia resulting from chronic disease (e.g., due to cancer, HIV/AIDS, rheumatoid arthritis, kidney disease, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), and other chronic inflammatory diseases), ulcers (such as a bleeding ulcer), myelodysplasia from cancer treatment (e.g., due to chemotherapy and/or radiation), iron deficiency, vitamin deficiency or an inability of process vitamin B-12, aplastic anemia (e.g., due to infection, medication, autoimmune disease, or exposure to toxic chemicals), bone marrow disease (e.g., due to leukemia and myelofibrosis), hemolytic anemia, sickle cell disease, thalassemia, or malaria.

In some examples, the subject has or is at risk of acquiring disorders or is undergoing drug treatments characterized by hemolysis. In some examples, the subject has or is at risk of hemorrhage. In some examples, the subject has or is at risk of exposure to radiation, such as at nuclear reactor accidents/meltdowns or during war. In some examples, the subject is at risk of hemorrhage due to an injury experience at a trauma scene, such as a car accident, suicide bombing, terrorist attack, bombing, or during war. For example, the disclosed methods include administering an effective amount of copeptin in the field and help people survive injuries including exposure to radiation or mass bleeding that otherwise would be lethal. Also, in irradiation injury, copeptin can be administered to enhance red blood cell manufacturing from quiescent progenitors that are much less affected by radiation.

Thus, in some examples, the method includes selecting a subject, such as selecting a subject at risk of acquiring or having anemia (for example due to hemorrhage or chemotherapy), prior to administering the effective amount of copeptin.

In some examples, the subject has a chronic disease (e.g., cancer, HIV/AIDS, rheumatoid arthritis, kidney disease, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), and other chronic inflammatory diseases), and ulcer (such as a bleeding ulcer), myelodysplasia from cancer treatment (e.g., due to chemotherapy and/or radiation), iron deficiency, vitamin deficiency (e.g. , folate and/or B-12) or an inability of process vitamin B-12), aplastic anemia (e.g., due to infection, medication, autoimmune disease, or exposure to toxic chemicals), bone marrow disease (e.g. , leukemia and myelofibrosis), hemolytic anemia, sickle cell disease, thalassemia, or malaria.

In some examples, the subject has a tumor, such as a cancer, and has previously received, will receive, or is receiving chemotherapy (such as radiation) treatment for the tumor. Thus, in some examples, the subject to be treated has a solid or liquid tumor. Exemplary solid include, such as breast carcinomas (e.g. lobular and duct carcinomas), sarcomas, carcinomas of the lung (e.g., non-small cell carcinoma, large cell carcinoma, squamous carcinoma, and adenocarcinoma), mesothelioma of the lung, colorectal adenocarcinoma, stomach carcinoma, prostatic

adenocarcinoma, ovarian carcinoma (such as serous cystadenocarcinoma and mucinous cystadenocarcinoma), ovarian germ cell tumors, testicular carcinomas and germ cell tumors, pancreatic adenocarcinoma, biliary adenocarcinoma, hepatocellular carcinoma, bladder carcinoma (including, for instance, transitional cell carcinoma, adenocarcinoma, and squamous carcinoma), renal cell adenocarcinoma, endometrial carcinomas (including, e.g., adenocarcinomas and mixed Mullerian tumors (carcinosarcomas)), carcinomas of the endocervix, ectocervix, and vagina (such as adenocarcinoma and squamous carcinoma of each of same), tumors of the skin (e.g., squamous cell carcinoma, basal cell carcinoma, malignant melanoma, skin appendage tumors, Kaposi sarcoma, cutaneous lymphoma, skin adnexal tumors and various types of sarcomas and Merkel cell carcinoma), esophageal carcinoma, carcinomas of the nasopharynx and oropharynx (including squamous carcinoma and adenocarcinomas of same), salivary gland carcinomas, brain and central nervous system tumors (including, for example, tumors of glial, neuronal, and meningeal origin), tumors of peripheral nerve, soft tissue sarcomas and sarcomas of bone and cartilage, and lymphatic tumors (including B-cell and T- cell malignant lymphoma).. In one example, the tumor is an adenocarcinoma. Exemplary liquid tumors include a lymphatic, white blood cell, or other type of leukemia. In a specific example, the subject has a tumor of the blood, such as a leukemia (for example acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia , and adult T-cell leukemia), lymphomas (such as Hodgkin’s lymphoma and non- Hodgkin’ s lymphoma), or myelomas).

In some examples, the subject is receiving, will receive, or has received chemotherapy, such as radiotherapy or chemoembolization. Exemplary chemotherapy agents include anti-tumor pharmaceutical treatments, such as anti-neoplastic chemotherapeutic agents, antibiotics, alkylating agents and antioxidants, kinase inhibitors, and other agents. Particular examples of chemotherapy agents include microtubule binding agents, DNA intercalators or cross-linkers, DNA synthesis inhibitors, DNA and/or RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors.

In some examples, the subject is receiving, will receive, or has received a microtubule binding agent. Such agents interact with tubulin to stabilize or destabilize microtubule formation thereby inhibiting cell division. Examples of microtubule binding agents include, without limitation, paclitaxel, docetaxel, vinblastine, vindesine, vinorelbine (navelbine), the epothilones, colchicine, dolastatin 15, nocodazole, podophyllotoxin and rhizoxin, as well as analogs and derivatives of such compounds. For example, suitable epothilones and epothilone analogs are described in International Publication No. WO 2004/018478. Taxoids, such as paclitaxel and docetaxel, as well as the analogs of paclitaxel taught by U.S. Patent Nos. 6,610,860; 5,530,020; and 5,912,264.

In some examples, the subject is receiving, will receive, or has received a DNA and/or RNA transcription regulator, such as actinomycin D, daunorubicin, doxorubicin and derivatives and analogs thereof.

In some examples, the subject is receiving, will receive, or has received a DNA intercalator or cross-linking agent, such as cisplatin, carboplatin, oxaliplatin, mitomycins, such as mitomycin C, bleomycin, chlorambucil, cyclophosphamide and derivatives and analogs thereof.

In some examples, the subject is receiving, will receive, or has received a DNA synthesis inhibitor, such as methotrexate, 5-fluoro-5'-deoxyuridine, 5-fluorouracil and analogs thereof.

In some examples, the subject is receiving, will receive, or has received an enzyme inhibitor, such as, camptothecin, etoposide, formestane, trichostatin and derivatives and analogs thereof.

In some examples, the subject is receiving, will receive, or has received a compound that affect gene regulation, such as an agent that results in increased or decreased expression of one or more genes, such as raloxifene, 5-azacytidine, 5-aza-2'-deoxycytidine, tamoxifen, 4- hydroxytamoxifen, mifepristone and derivatives and analogs thereof.

In some examples, the subject is receiving, will receive, or has received an angiogenesis inhibitor, such as angiostatin Kl-3, staurosporine, genistein, fumagillin, medroxyprogesterone, suramin, interferon- alpha, metalloproteinase inhibitors, platelet factor 4, somatostatin, thromobospondin, endostatin, thalidomide, and derivatives and analogs thereof.

In some examples, the subject is receiving, will receive, or has received a kinase inhibitor, such as Gleevac, Iressa, or Tarceva.

In some examples, the subject is receiving, will receive, or has received a therapeutic antibody, such as a monoclonal antibody, such as alemtuzumab, atezolizumab, bevacizumab, blinatumomab, cetuximab, olaratumab, rituximab, or trastuzumab.

In some examples, the subject is receiving, will receive, or has received adriamycin, apigenin, rapamycin, zebularine, cimetidine, and derivatives and analogs thereof.

In some examples, the subject is receiving, will receive, or has received interleukin-2 (IL-2), anti-CTLA-4, anti-PDl, or combinations thereof.

In some examples, the disclosed methods are used to treat polycythemia. For example, copeptin, or an agonist or an inhibitor thereof, can be used to treat polycythemia. In some examples, copeptin inhibits development and decreases numbers of HSCs, thus decreasing the number of circulating cells and diluting the blood. Thus, in some examples the subject treated has or is at risk for polycythemia.

In some examples, the disclosed methods are used to treat an autoimmune disease. For example, copeptin can be used to modulate an immune response. In some examples, copeptin regulates T cell formation, B cell formation, or both. Thus, in some examples, the subject treated with the disclosed methods has an autoimmune disease, such as encephalomyelitis (EAE), lupus, rheumatoid arthritis, celiac disease, Sjogren's syndrome, polymyalgia rheumatic, multiple sclerosis, ankylosing spondylitis, alopecia areata, vasculitis or temporal arteritis.

In some examples, the disclosed methods are used to treat diabetes. A composition comprising a therapeutically effective amount of AVP and a therapeutically effective amount of copeptin can be used to treat a diabetes. Thus, in some examples, the subject treated with the disclosed methods has central diabetes insipidus, or is at risk of developing diabetes (e.g., pre diabetic).

In some examples, the disclosed methods are used to stimulate bone, cartilage or both. In some examples, copeptin can be used to stimulate bone or cartilage formation, or both bone and cartilage formation. Thus, in some examples, the subject treated with the disclosed methods has or is at risk of developing a fracture, or has or may be at risk of developing osteoporosis. Stimulating bone may include increasing bone mass density in the subject. The subject may have, may have had or may be at risk of, requiring knee, hip, or shoulder, replacement surgery.

Additional Therapeutic Molecules

In some examples, the method includes the use of additional agents in combination with copeptin.

In some examples, the method includes administering an effective amount of the copeptin and one or more additional therapeutic agents, such as an effective amount of one or more erythropoiesis modulatory molecules, such as an arginine vasopressin (A VP), arginine vasopressin receptor 1B (AVPR1B) agonist(s), arginine vasopressin receptor 1A (AVPR1A) agonist, erythropoietin, or any combination thereof. In some examples, the additional therapeutic agent is administered at least one hour after the administration of the copeptin, such at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 96 hours after, such as between 2 and 96 hours, 12 and 72 hours, 24 and 48 hours, including 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 18 hours, 24 hours, 30 hours 36 hours, 48 hours, 72 hours, or 96 hours after. In some examples, the additional therapeutic agent is administered at least one hour before the administration of the copeptin, such at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 96 hours after, such as between 2 and 96 hours, 12 and 72 hours, 24 and 48 hours, including 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 18 hours, 24 hours, 30 hours 36 hours, 48 hours, 72 hours, or 96 hours before. In some examples, the additional therapeutic agent is administered simultaneously or contemporaneously with the copeptin.

In some examples, the method includes administering an effective amount of one or more erythropoiesis modulatory molecules, such as AVP, AVPR1B agonist(s), EPO, or any combination thereof, prior to administering an effective amount of copeptin. Thus, in some examples, the additional therapeutic agent is administered at least one hour before the administration of the copeptin, such at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 96 hours before, such as between 2 and 96 hours, 12 and 72 hours, 24 and 48 hours, including 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 18 hours, 24 hours, 30 hours 36 hours, 48 hours, 72 hours, or 96 hours before. In some examples, the additional therapeutic agent is administered at least one hour before the administration of the copeptin, such at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 96 hours after, such as between 2 and 96 hours, 12 and 72 hours, 24 and 48 hours, including 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 18 hours, 24 hours, 30 hours 36 hours, 48 hours, 72 hours, or 96 hours before. In some examples, the additional therapeutic agent is administered simultaneously or contemporaneously with the copeptin.

In some examples, the effective amount copeptin and one or more additional therapeutic agents, such as an effective amount of one or more erythropoiesis modulatory (e.g., stimulatory) molecules, such as a therapeutically effective amount of AVP, AVPR1B agonist(s), EPO, or any combination thereof, are administered together or contemporaneously.

In one example, the additional agent includes EPO or one or more AVP receptor agonists, such as an AVPR1A and/or AVPR1B agonist. Examples of such molecules include one or more of D[Leu ⁴ , Lys ⁸ ]-vasopressin, dCha ⁴ AVP, [Arg ⁸ ]-vasopressin (AVP), [Phe2, Ile3, Om8] vasopressin, (Deamino-Cysl,Val ⁴ ,D-Arg ⁸ )-vasopressin, as well as agents disclosed in Manning et al. { J .

Neuroendocrinology 24:609-628, 2012), Manning et al. {Prog. Brain Res. 170: 473-512, 2008) and Craighead et al. {Prog. Brain Res. 170: 527-535, 2008) each of which is hereby incorporated by reference in its entirety. In some examples, the AVPR1B receptor agonist is peripherally active (/. _<? ., the agonist has no effect on the central nervous system).

In some examples, the additional agent includes one or more erythropoiesis modulatory molecules, such as erythropoietin, including erythropoietins available for use as therapeutic agents and produced by recombinant DNA technology in cell culture, alone or in combination with a therapeutically effective amount of AVP, AVPR1B agonist(s), or a combination thereof. Examples include Epogen/Procrit (epoetin alfa) and Aranesp (darbepoetin alfa). In some examples, the one or more additional erythropoiesis stimulatory molecules include one or more recombinant EPO including one or more of epoetin alfa (Darbepoetin (Aranesp); Epocept (Lupin pharma); Nanokine (Nanogen Pharmaceutical biotechnology); Epofit (Intas pharma); Epogen (Amgen); Epogin; Eprex, (Janssen-Cilag); Binocrit (Sandoz; Procrit), epoetin beta (NeoRecormon, made by Hoffmann-La Roche; Recormon; Methoxy polyethylene glycol-epoetin beta (Mircera) by Roche), epoetin delta (Dynepo trademark name for an erythropoiesis stimulating protein, by Shire plc), epoetin omega: (Epomax), epoetin zeta ((biosimilar forms for epoetin alpha): Silapo (Stada); Retacrit (Hospira)), EPOTrust (made by Panacea Biotec Ltd), Erypro Safe (made by Biocon Ltd.), Repoitin (Seram Institute of India Limited) Vintor (Emcure Pharmaceuticals); Erykine (Intas Biopharmaceutica), Wepox (Wockhardt Biotech), Espogen (LG life sciences), ReliPoietin (Reliance Life Sciences), Shanpoietin (Shantha Biotechnics Ltd), Zyrop (Cadila Healthcare Ltd.), EPIAO (rHuEPO, made by Shenyang Sunshine Pharmaceutical Co.. LTD. China), NESP (Macdougall IC (July 2000) "Novel erythropoiesis stimulating protein" Semin. Nephrol. 20 (4): 375-81. PMID 10928340), or erythropoiesis stimulating molecules disclosed in Craighead et al. (Prog. Brain Res. 170: 527-535, 2008), Guillon et al. ( J . Neuroendocrinol. 16(4): 356-361, 2004), Manning et al. (J.

Neuroendocrinology 24:609-628, 2012), and Baker et al , Bioorg Med Chem Lett 2l(l2):3603- 3607, 2011 (all hereby incorporated by reference in their entirety).

In some examples, the additional agent includes one or more therapeutic agents that increase bone density, such as one or more of bisphosphonates, calcitonin, denosumab, teriparatide and raloxifene.

In some examples, the additional agent includes one or more therapeutic agents used to treat an autoimmune disease, such as one or more of methylprednisolone, kenalog, Medrol,

prednisolone, Solu-Medrol, hydrocortisone, Cortef, triamcinolone acetonide, cortisone, Celestone Soluspan, Orapred, betamethasone acetate and sodium phosphate, Veripred, Pediapred, Millipred and dexamethasone. In some examples, the copeptin and/or one or more additional therapeutic agents are linked to one or more immunoglobulin Lc domains to improve the therapeutic effect of the copeptin or the one or more therapeutic agents, increase their plasma half-life or improve their solubility and/or stability. Immunoglobulin Lc domains are known, and one of ordinary skill in the art can identify binding partners and attach the copeptin or one or more therapeutic agents to one or more Lc domains.

Exemplary Administration

Lor any of the disclosed methods, an effective amount of copeptin is one when administered by a particular route and concentration, induces the desired response (e.g., stimulating HSC cell proliferation and/or differentiation, treatment of anemia, treatment of hemorrhaging, and the like).

Copeptin and other therapeutic molecules can be administered topically (e.g., in a patch), nasally, injection (e.g., intravenously, intramuscularly, parenterally, intraosseous, intratumorally), orally, rectally, vaginally, or as implants, or using a combination of these techniques. In some examples, copeptin or other therapeutic agent is injected.

Suitable solid or liquid pharmaceutical preparation forms of copeptin and other therapeutic molecules include aerosols, (micro)capsules, creams, drops, drops or injectable solution in ampoule form, emulsions, granules, powders, suppositories, suspensions, syrups, tablets, coated tablets, and also preparations with protracted release of copeptin and other therapeutic molecules (such as in a skin patch), in whose preparation excipients and additives and/or auxiliaries such as binders, coating agents, disintegrants, flavorings, lubricants, solubilizers, sweeteners, or swelling agents. Copeptin and other therapeutic molecules are suitable for use in a variety of drug delivery systems (e.g., see Langer,“New Methods of Drug Delivery,” Science 249: 1527-1533, 1990).

Copeptin and other therapeutic molecules can be formulated into therapeutically-active pharmaceutical agents that can be administered to a subject parenterally or orally. Parenteral administration routes include, but are not limited to epidermal, intraarterial, intramuscular (IM and depot IM), intraperitoneal (IP), intravenous (IV), intraosseous, intratumoral, intrasternal injection or infusion techniques, intranasal (inhalation), intrathecal, injection into the stomach, subcutaneous injections (subcutaneous (SQ and depot SQ), transdermal, topical, and ophthalmic.

Copeptin and other therapeutic molecules can be mixed or combined with suitable pharmaceutically acceptable excipients to prepare pharmaceutical agents. Pharmaceutically acceptable excipients include, but are not limited to, alumina, aluminum stearate, buffers (such as phosphates), glycine, ion exchangers (such as to help control release of charged substances), lecithin, partial glyceride mixtures of saturated vegetable fatty acids, potassium sorbate, serum proteins (such as human serum albumin), sorbic acid, water, salts or electrolytes such as cellulose- based substances, colloidal silica, disodium hydrogen phosphate, magnesium trisilicate, polyacrylates, polyalkylene glycols, such as polyethylene glycol, polyethylene-polyoxypropylene- block polymers, polyvinyl pyrrolidone, potassium hydrogen phosphate, protamine sulfate, group 1 halide salts such as sodium chloride, sodium carboxymethylcellulose, waxes, wool fat, and zinc salts, for example. Liposomal suspensions may also be suitable as pharmaceutically acceptable carriers.

Upon mixing or addition of copeptin and/or other therapeutic molecules with an appropriate carrier, the resulting mixture may be a solid, solution, suspension, emulsion, or the like.

Pharmaceutical carriers suitable for administration of copeptin and/or other therapeutic molecules include any such carriers known to be suitable for the particular mode of administration. In addition, copeptin or other therapeutic substance can also be mixed with other inactive or active materials that do not impair the desired action, or with materials that supplement the desired action, or have another action.

Methods for solubilizing copeptin or other therapeutic substance may be used where the agents exhibit insufficient solubility in a carrier. Such methods include, but are not limited to, dissolution in aqueous sodium bicarbonate, using cosolvents such as dimethylsulfoxide (DMSO), and using surfactants such as TWEEN® (ICI Americas, Inc., Wilmington, DE).

Copeptin or other therapeutic agent can be prepared with carriers that protect them against rapid elimination from the body, such as coatings or time-release formulations. Such carriers include controlled release formulations, such as, but not limited to, microencapsulated delivery systems. Copeptin or other therapeutic agent is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect, typically in an amount to avoid undesired side effects, on the treated subject. The therapeutically effective concentration may be determined empirically by testing the compounds in in vitro and in vivo model systems for the treated condition. For example, mouse or rat models may be used to determine effective amounts or concentrations that can then be translated to other subjects, such as humans.

Injectable solutions or suspensions of copeptin or other therapeutic agent can be formulated, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as l,3-butanediol, isotonic sodium chloride solution, mannitol, Ringer’s solution, saline solution, or water; or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid; a naturally occurring vegetable oil such as coconut oil, cottonseed oil, peanut oil, sesame oil, and the like; glycerin; polyethylene glycol; propylene glycol; or other synthetic solvent; antimicrobial agents such as benzyl alcohol and methyl parabens; antioxidants such as ascorbic acid and sodium bisulfite; buffers such as acetates, citrates, and phosphates; chelating agents such as ethylenediaminetetraacetic acid (EDTA); agents for the adjustment of tonicity such as sodium chloride and dextrose; and combinations thereof. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass, plastic, or other suitable material. Buffers, preservatives, antioxidants, and the like can be incorporated as required. Where administered intravenously, suitable carriers include physiological saline, phosphate-buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropyleneglycol, and mixtures thereof. Liposomal suspensions, including tissue-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers.

For topical application, copeptin or other therapeutic agent may be made up into a cream, lotion, ointment, solution, or suspension in a suitable aqueous or non-aqueous carrier. Topical application can also be accomplished by transdermal patches or bandages, which include the therapeutic substance. Additives can also be included, e.g., buffers such as sodium metabisulphite or disodium edetate; preservatives such as bactericidal and fungicidal agents, including phenyl mercuric acetate or nitrate, benzalkonium chloride, or chlorhexidine; and thickening agents, such as hypromellose.

If copeptin, or other therapeutic agent, is administered orally as a suspension, the pharmaceutical formulation may contain a suspending agent, such as alginic acid or sodium alginate, bulking agent, such as microcrystalline cellulose, a viscosity enhancer, such as methylcellulose, and sweeteners/flavoring agents. Oral liquid preparations can contain

conventional additives such as suspending agents, e.g., gelatin, glucose syrup, hydrogenated edible fats, methyl cellulose, sorbitol, and syrup; emulsifying agents, e.g., acacia, lecithin, or sorbitan monooleate; non-aqueous carriers (including edible oils), e.g., almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives such as methyl or propyl p-hydroxybenzoate or sorbic acid; and, if desired, conventional flavoring or coloring agents. When formulated as immediate release tablets, these agents can contain dicalcium phosphate, lactose, magnesium stearate, microcrystalline cellulose, and starch and/or other binders, diluents, disintegrants, excipients, extenders, and lubricants.

If oral administration is utilized, copeptin or other therapeutic agent can be provided in a composition that protects it from the acidic environment of the stomach. For example, copeptin or other therapeutic agent can be formulated with an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. Copeptin or other therapeutic agent can also be formulated in combination with an antacid or other such ingredient.

Oral compositions generally include an inert diluent or an edible carrier and can be compressed into tablets or enclosed in gelatin capsules. For the purpose of oral therapeutic administration, copeptin or other therapeutic agent can be incorporated with excipients and used in the form of capsules, tablets, or troches. Pharmaceutically compatible adjuvant materials or binding agents can be included as part of the composition. The capsules, pills, tablets, troches, and the like can contain any of the following ingredients or compounds of a similar nature: a binder such as, but not limited to, acacia, corn starch, gelatin, gum tragacanth, polyvinylpyrrolidone, or sorbitol; a filler such as calcium phosphate, glycine, lactose, microcrystalline cellulose, or starch; a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate, polyethylene glycol, silica, or talc; a gildant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; disintegrants such as potato starch; dispersing or wetting agents such as sodium lauryl sulfate; and a flavoring agent such as peppermint, methyl salicylate, or fruit flavoring.

When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier, such as a fatty oil. In addition, dosage unit forms can contain various other materials that modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. One or more of the disclosed modulatory agents, or other therapeutic agent can also be administered as a component of an elixir, suspension, syrup, wafer, tea, chewing gum, or the like. A syrup may contain, in addition to the active compounds, sucrose or glycerin as a sweetening agent and certain preservatives, dyes and colorings, and flavors.

When administered orally, copeptin or other therapeutic agent can be administered in usual dosage forms for oral administration. These dosage forms include the usual solid unit dosage forms of tablets and capsules as well as liquid dosage forms such as solutions, suspensions, and elixirs. When the solid dosage forms are used, they can be of the sustained release type so that the compounds need to be administered less frequently.

The subject treated with the disclosed methods is typically treated for a sufficient period of time to reduce, inhibit or prevent one or more signs or symptoms that would benefit from hematopoiesis, such as anemia. For example, the subject may be treated for at least 1 day, at least 2 days, at least 3 days, at least 4 days at least 5, days, at least 6 days, at least 7 days, at least 10 days, at least 14 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 6 months, at least 1 year, or longer, such as for 1 day, 2 days, 3 days, 4 days 5, days, 6 days, 7 days,

10 days, 14 days, 1 month, 2 months, 3 months, 4 months, 6 months, 1 year, 2 years, 3 years, 4 years, or 65 years, such as for 2 to 96 hours, for 12 to 72 hours, or for 24 to 48 hours. In some embodiments, the effective amount of copeptin is administered as a single dose per time period, such as every 6 hours, every 12 hours, daily, weekly, or monthly. Copeptin can also be

administered in several doses intermittently, such as every few days (for example, at least about every two, three, four, five, or ten days) or every few weeks (for example at least about every two, three, four, five, or ten weeks).

Treatment can be initiated prior to the on-set of a particular condition, such as anemia, to a subject at risk of acquiring anemia, such as a subject undergoing chemotherapy. In some examples, the treatment with copeptin is initiated before chemotherapy treatment, such as at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 6 hours, at least 8 hours, at last 12 hours, at least 24 hours, at least 48 hours, or at least 72 hours, before chemotherapy is administered. In some examples, the treatment with copeptin is initiated after chemotherapy treatment, such as at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 6 hours, at least 8 hours, at last 12 hours, at least 24 hours, at least 48 hours, or at least 72 hours, after chemotherapy is administered. In some examples, copeptin is administered before and after chemotherapy treatment.

In some cases, a single administration of copeptin is effective to provide the desired therapeutic effect. In further examples, additional administrations are provided to achieve the desired therapeutic effect.

Amounts effective for various therapeutic treatments of the present disclosure may depend on the severity of the condition/disease and the weight and general state of the subject, as well as the absorption, inactivation, and excretion rates of the copeptin, the dosage schedule, and amount administered, as well as other factors. Dosages used in vitro or in an animal model can provide useful guidance in the amounts useful for in vivo administration of copeptin. For example, mouse or rat models can be used to determine effective dosages that can then be translated to dosage amount for other subjects, such as humans. Various considerations in dosage determination are described, e.g., in Gilman el al. , eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press (1990); and Remington's Pharmaceutical Sciences, l7th ed., Mack Publishing Co., Easton, Pa. (1990).

Particular dosage regimens can be tailored to a particular subject, condition to be treated or desired result. For example, an initial treatment regimen with copeptin can be applied to arrest or slow the condition. Such initial treatment regimen may include administering a higher dosage of copeptin, or administering such material more frequently, such as daily. After a desired therapeutic result has been obtained, such as a desired level of HSC proliferation, a second treatment regimen may be applied, such as administering a lower dosage of copeptin or administering copeptin less frequently, such as monthly, bi-monthly, quarterly, or semi-annually. In such cases, the second regimen may serve as a“booster” to restore or maintain a desired level of HSC proliferation. In specific examples, copeptin is administered to a subject in an amount sufficient to provide a dose of copeptin of between about 10 nmol/g and about 500 nmol/g, such as between about 2 nmol/g and about 20 nmol/g or between about 2 nmol/g and about 10 nmol/g. In some examples, copeptin is administered to a subject in an amount sufficient to provide a dose of about 0.01 pg/kg to about 1000 mg/kg, about 1 pg/kg to about 1000 pg/kg, about 10 ng/kg to about 500 pg/kg, about 50 pg/kg to about 500 pg/kg, or about 0.1 mg/kg to about 1000 mg/kg; in particular examples this amount is provided per day or per week. In another example, copeptin is

administered to a subject in an amount sufficient to provide a dose of copeptin of about 0.1 mg/kg to about 1 mg/kg. In further examples, copeptin is administered to a subject in an amount sufficient to provide a concentration of copeptin in the administrated material of about 5 nM to about 500 nM, such as about 50 nM to about 200 nm, or about 100 nM. In other examples, copeptin is

administered to a subject at about 500 pg/ml to about 1 pg/ml, such as about 300 pg/ml to about 3 pg/ml, about 200 pg/ml to about 20 pg/ml, including 500 pg/ml, 400 pg/ml, 300 pg/ml, 250 pg/ml, 200 pg/ml, 150 pg/ml, 100 pg/ml, 50 pg/ml, 25 pg/ml, 12.5 pg/ml, 6.25 pg/ml, 3.125 pg/ml, 2.5 pg/ml or 1.25 pg/ml.

In some examples, the specific concentration of copeptin administered is determined based upon receptor affinity and bioavailability.

In some examples, the copeptin and/or one or more additional therapeutic agents are linked to one or more immunoglobulin Fc domains and administered as Fc-fusion proteins to improve the therapeutic effect of the copeptin or the one or more therapeutic agents, increase their plasma half- life or improve their solubility and/or stability. Immunoglobulin Fc domains are known in the art and publicly available, and one of ordinary skill in the art can identify binding partners and attach the copeptin or one or more therapeutic agents to one or more Fc domains.

Copeptin-Containing Compositions

Also provided are compositions that include copeptin and one or more additional therapeutic agents, such as one or more erythropoiesis stimulatory molecules (such as EPO), one or more lymphopoiesis stimulatory molecules, one or more erythropoiesis inhibitory molecules, one or more lymphopoiesis inhibitory molecules, one or more immunomodulatory molecules, one or more bone and/or cartilage stimulatory molecules, or combinations thereof. The active ingredients in the composition can be present in therapeutically effective amounts. Exemplary chemotherapeutic agents, erythropoiesis stimulatory molecules and lymphopoiesis stimulatory molecules, which can be included in the composition, are provided above. Such compositions can also include a physiologically acceptable carrier, such as one or more of those provided above.

In some examples, such a composition is present in a vial or container, such as a glass or plastic container. In some example, such a composition is part of a pharmaceutical composition, such as an injectable solution, lyophilized composition, patch, tablet, capsule or suppository.

Example 1

Effect of Copeptin on Steady-State Erythropoiesis

To demonstrate the effect of copeptin in steady-state erythropoiesis (e.g., in subject who has normal levels of blood cells, and who may not need to have increased cell production in the bone marrow), mice were injected with copeptin or PBS for 6 consecutive days at a dose of 100 pg/kg body weight. The mice are analyzed on the 7 ^th day. PBS was used as a control. An overview of the method is shown in FIG. 2A.

As steady-state erythropoiesis is primarily restricted to bone marrow (BM), cellularity (the density/number of mononuclear blood cells) and the different blood lineage cells in the BM were examined. Total bone marrow cellularity was significantly increased in copeptin injected mice compared to vehicle injected animals (FIG. 2B). Erythroid cells (FIG. 2C) were significantly downregulated, while myeloid cells (FIG. 2D) were significantly upregulated in copeptin injected mice compared to control mice. B-cells and T-cells (FIGS. 2E., 2F) did not change in copeptin injected mice compared to control mice, indicating that other populations (monocyte/macrophage and/or the granulocyte) are responsible for of the increase. Lineage cells in the spleen were not affected by copeptin administration (FIGS. 3A-3D).

Erythroid precursors were examined at various stages of maturation using flow cytometry (FIG. 4A). Copeptin injected mice had significantly lower numbers of orthochromatic erythroblasts and reticulocytes compared to control mice (FIG. 4B), but their RBC in the BM were significantly higher. The peripheral blood did not have increased numbers of RBCs (Terl 19 positive cells) at the same timepoint (FIG. 5A) and there was no change in the number of circulating reticulocytes (FIG. 5B).

As copeptin affected erythroid precursors in the bone marrow, using a methylcellulose assay, it was determined whether copeptin has an action on the progenitor cells. As shown in FIG. 6, the erythroid progenitors, BFU-E, and multipotent progenitors, CFU-GEMM were significantly down-regulated in copeptin injected mice as compared to control mice. Myeloid progenitors, CFU- GM, were unchanged. Thus, copeptin may regulate multipotent and erythroid progenitors.

These results indicate that copeptin has an inhibitory effect on erythroid progenitors and precursors in steady state erythropoiesis (FIG. 7).

Example 2

Effect of Copeptin on Stress Erythropoiesis

To demonstrate the effect of copeptin on stress induced erythropoiesis (e.g., in subject who has reduced levels of blood cells, and who may need to have increased cell production in the bone marrow), a phenylhydrazine (PHZ)-induced model of anemia was used. Injecting PHZ causes anemia by lysing the membrane of red blood cells. Mice were injected for 7 days with copeptin (100 pg/kg body weight). PHZ (60 mg/kg) was injected once (after four days of copeptin administration). The mice were analyzed 3 days later (e.g., analysis of cells in the bone marrow and spleen). An overview is shown in FIG. 8 A.

In the bone marrow, polychromatic and orthochromatic erythroblasts were significantly increased in copeptin injected mice vs controls (FIG. 8B). In the spleen, which is the primary organ where stress hematopoiesis occurs in mice, all the erythroid precursor populations were

significantly upregulated in copeptin injected mice compared to controls, but RBCs were not (FIG. 8C). The reticulocyte count in the peripheral blood (FIG. 9) increased in copeptin-treated mice. There were no significant differences in progenitor levels in the bone marrow (FIG. 10A) or spleen (FIG. 10B) in copeptin as compared to control mice.

These results indicate that copeptin promotes stress erythropoiesis by an effect on RBC precursors.

Example 3

Effect of Copeptin on Anemia

The effect of copeptin in mouse anemia models can be examined as follows. 8- to ^-week- old age- and gender-matched C57BL/6 mice are used.

In one example, the anemia model is a hemorrhage model. In this model, 25% of the circulating blood volume (based on Animal Research Advisory Committee Guidelines for survival of mice and rats after blood loss) will be drained from the retro-orbital plexus. In one example, the anemia model is a sublethal irradiation model. In this model, mice will be irradiated (4.5 Grays in a single dose).

After hemorrhage or irradiation, mice are injected with different doses of copeptin, such as about 10 ng/kg to 500 pg/kg (such as 100 pg/kg). Blood samples will be taken on day 2, 4, and in some cases, day 6, and hematocrit and corrected reticulocyte values determined. The reticulocyte ratio in peripheral blood can be determined using the Retic-Count flow kit (BD Biosciences) according to the manufacturer's instructions. Hematocrit values can be measured with an automated analyzer and microhematocrit tubes. The reticulocyte index will be calculated as follows:

reticulocyte ratio % /total gated cells x hematocrit /45. Bone marrow and spleen will be analyzed (e.g., every other day for one week, or for a long term treatment, the copeptin can be administered using a pump (e.g. , Alzet minipump at a constant dose (e.g., 10 ng/kg to 500 ug/kg) for 3-4 weeks to observe chronic effects. Methylcellulose assay and flow cytometry can be used to analyze bone marrow and spleen cells as described below.

Spleen is the organ of stress hematopoiesis in mice. To demonstrate that copeptin induces erythropoiesis independent of the spleen, splenectomized mice (C57 black male mice about 6-12 weeks of age) are used. To perform splenectomies, mice are anesthetized with isoflurane, and a 2 cm dorsal midline skin incision made with its caudal terminus at the level of the l3th rib. The spleen will be exposed by opening the left abdominal wall 1.5 to 2.5 cm from midline. With blunt forceps, the organ (with accompanying blood vessels and pancreatic tissue) pulled through the incision. After ligating the blood vessels the spleen will be removed, and the skin incision closed with wound clips. Sham splenectomies are performed identically except that no ligation takes place and the spleen will be intact. These mice will be analyzed in a manner similar to the above mice. Briefly, splenectomized mice are injected with different doses of copeptin, such as doses in the range of about 10 ng/kg to 500 ug/kg (such as 100 mg/kg), or with vehicle control, and blood samples and bone marrow samples subsequently analyzed for hematopoiesis, for example using flow cytometry.

Example 4

Role of copeptin on human hematopoietic stem and progenitors

Human CD34+ cells (human hematopoietic stem cells) can be isolated from peripheral blood huffy coats from healthy volunteers. These cells can be cultured in Stemspan medium with penicillin and streptomycin, supplemented with 100 ng/ml thrombopoietin (TPO), 50 ng/ml Fms- related tyrosine kinase 3 ligand (FLT3L), 50 ng/ml IL-3, and 100 ng/ml stem cell factor (SCF) (Peprotech). Cells are treated with different doses of copeptin, such as about 10 ng/kg to 500 pg/kg (such as 100 pg/kg). After 24 hours, BrdU at a 1: 1000 concentration is added to the cultures and BrdU incorporation measured, for example using the Cell Proliferation ELISA BrdU kit (Roche) after another 24 hours of incubation. The expanded cells can be used for methycellulose assay as described below.

Human CD34+ cells are cultured in a two-phase liquid culture to erythrocytes. Briefly, phase I is for 6 days in phase I medium containing Stemspan medium with 10% FBS, 100 ng/ml stem cell factor (SCF), 10 ng/ml interleukin 3 (IL-3), 0.5 U/ml EPO, 10 ⁵ M b-mercapthoethanol, penicillin, and streptomycin (37°C, 5% CO2). In phase II, cells are cultured in Stemspan medium with 30% FBS, 10 ⁵ M b-mercapthoethanol, 10 ng/ml SCF, 2 U/ml EPO, and antibiotics for eight days. Copeptin at different doses (such as 10 ng/kg to 500 ug/kg) can be used throughout the culture or only during phase I or phase II. Cells will be counted at the indicated time points for growth curve analysis and cytospined cells will be morphologically analyzed.

The following methods can be used to analyze human and mice hematopoiesis in vitro and in vivo, respectively.

Methylcellulose assay

Human CD34 ⁺ cells or mouse bone marrow cells are plated into StemMACS HSC-CFU basic medium (Miltenyi Biotech) containing hematopoietic factors (granulocyte-macrophage colony-stimulatory factor (GM-CSF) 5 ng/ml, IL-3 5 ng/ml, SCF 50 ng/ml, and EPO 2.5 U/ml) for BFU-E assays and counted on day 14. For CFU-E assays, cells are grown in StemMACS HSC- CFU basic medium with 3 U/ml of EPO and counted on day 7. Copeptin is added to the media at different concentrations (such as 10 ng/kg to 500 ug/kg). For pre-treatment experiments, CD34+ cells are stimulated with copeptin in Stemspan medium supplemented with 100 ng/ml TPO, 50 ng/ml FLT3L, 50 ng/ml IL-3, and SCF 100 ng/ml for 48 hours. After 48 hours, the cells will be seeded in StemMACS HSC-CFU basic medium with cytokines as described above. Colonies are counted, for example using a STEMvision automated CFU colony counter (STEMCELL

Technologies). Flow cytometry

Mouse erythroid lineage cells are FACS sorted using CD44 and Terll9. Briefly, 4-month- old C57BL/6 mouse bone marrow is extracted and CD45+ cells depleted. CD45- cells are sorted based on CD1 lb-, Grl-, and Terl 19 into four populations. For mouse hematopoietic stem cells, lineage negative, Sca-l positive, and c-kit positive (LSK) cells are sorted. Rat anti-mouse lineage antibodies (anti-CD4, CD8, B220, Terl l9, Gr-l, CDllb) are used for lineage depletion. Lineage negative cells are stained with c-kit PE/Cy5, and Sca-l-FITC antibodies and LSK cells are sorted, for example on a DAKO Cytomation MoFlo cell sorter.

Example 5

Treatment of Polycythemia

This example provides methods that can be used to treat polycythemia using therapeutic amounts of copeptin (or an agonist or antagonist thereof). In some examples, busulfan and/or hydroxyurea are also administered. In some examples, the subject is also treated with one or more phlebotomies to reduce the number of RBCs in the blood. For example, therapeutic amounts of copeptin (or an antagonist thereof) can be used to reduce the number of RBCs produced, or the number in the blood. Polycythemia occurs when too many cells are released into the circulation from the bone marrow. When too many red cells circulate, the blood becomes very viscous (thick) and there is danger of blocking blood vessels (emboli). Copeptin may regulate the last phase of red cell production and limit the number of RBCs that enucleate (exclude their nuclei) which is necessary for the cells to be able to circulate and carry oxygen. These nucleated cells do not get out into the circulation, thus the number of circulating cells will be decreased.

In one example, the subject has primary polycythemia (polycythemia vera), which is caused by an overproduction of red cells in the bone marrow. This is a disorder due to intrinsic problems with red cell precursors and the blood gets too“thick” which is represented by high hematocrit values. Copeptin (or an antagonist thereof) can be used to decrease the RBC production in the bone marrow.

In one example, the subject has secondary polycythemia, for example due to increased production of EPO or abnormalities in the EPO receptor.

A subject is administered a therapeutic amount of copeptin, such as a mouse at a dose of 10 ng/kg to 500 ug/kg, such as 100 pg/kg. In some examples, the subject is treated indefinitely, for example for life. In one example the subject is a mouse model for polycythemia, such as one with a mutated EPOR (e.g., see Divoky et al., PNAS 98:986-91, 2001).

Example 6

Inducing WBC Production

This example provides methods that can be increase or induce proliferation of WBCs using therapeutic amounts copeptin. For example, a subject having a low WBC count, such as one having a malignancy, toxicosis, chemotherapy damage, irradiation damage, infection in the bone marrow, or combinations thereof, resulting in a low white cell count can be treated with the methods provided herein. Copeptin might induce the progenitor or only subpopulations of white cells.

The normal white cell count is usually between 4 x l0 ⁹ /L and 11 x l0 ⁹ /L. In the US this is usually expressed as 4,000 to 11,000 white blood cells per microliter of blood. Thus, in some examples a subject treated with the disclosed methods have a white cell count of less than 4000 white cells/ml of blood.

A subject in need of increased WBC production (for example a subject with a reduced WBC count) is administered a therapeutic amount of copeptin, such as a mouse at a dose of 10 ng/kg to 500 ug/kg, such as 100 pg/kg.

Example 7

Treatment using EPO and Copeptin

This example provides methods that can be used to treat a disease, such as anemia, using a combination of therapeutic amounts of EPO and copeptin.

EPO is constantly released and stimulates hematopoietic stem cells to differentiate into proerythrocytes, which is the most undifferentiated red cell progenitor. This cell needs to go through divisions to reach the last stage of development (called orthohromatic erythrocyte) and this takes 3-5 days. Copeptin can be used to accelerate the development of the late stages of this process, thus release more mature cells very quickly (e.g., within 24 hours). Thus treatment with both EPO and copeptin can result in a continuous flow of new RBCs into the circulation, which can alleviate hypoxia (too little oxygen is carried by low number of RBCs to the organs) and anemia.

A subject in need of increased RBC production (for example a subject with anemia) is administered a therapeutic amount of copeptin, such as a mouse at a dose of 10 ng/kg to 500 ug/kg, such as 100 pg/kg, and a therapeutic amount of EPO (e.g., epoetin alpha), such as subcutaneous 150 units/kg 3 times weekly, or 40,000 units weekly; or IV/Subcutaneous 50 to 100 units/kg 3 times weekly.

Example 8

Effect of copeptin and vasopressin (A VP), AVPR1B agonist(s), EPO, and combinations thereof on HSC and progenitors

To demonstrate the role of AVP, AVPR1B agonists, EPO, or a combination thereof, and copeptin on bone marrow function in vitro, (separately as well as their synergistic effect), human CD34+ hematopoietic stem cells and mouse LSK hematopoietic stem cells are unstimulated or stimulated with different dosages of copeptin, AVP, AVPR1B agonist(s), EPO, or a combination thereof, at different times, and their effect is determined by colony forming assay, differentiation culture assay and flow cytometry analysis.

Human CD34+ cells are isolated and cultured as described in Example 4. LSK cells are isolated from the bone marrow of 4 to 8-week-old C57BL/6 (CD45.2) mice and B6.Ly5.2

(B6.Ly5 ^SIL )(CD45.l) mice by flushing the femur and tibia of mice with cold DMEM containing 2% FBS and 100 ng/ml penicillin/streptomycin. Thymus and spleen cell suspensions are prepared by mincing the organs through nylon mesh. Mononuclear cells are obtained after gradient

centrifugation using lymphocyte separation medium (Cellgro, Mediatech, VA). The LSK cells thus obtained are cultured in DMEM in the presence of Percoll (Amersham Biosciences Corp.) to adjust gravity, STEMPRO-34 Nutrient Supplement (Invitrogen, Carlsbad, CA) and cytokines.

Colony Forming Assay

Unstimulated and stimulated CD34+ cells or LSK cells are cultured in a semi-solid methylcellulose matrix supplemented with erythroid progenitor cells (CFU-erythroid [CFU-E], burst-forming unit-erythroid [BFU-E]), granulocyte and macrophage progenitor cells (CFU- granulocyte, macrophage [CFU-GM], multi-potential progenitor cells (CFU-granulocyte, erythrocyte, macrophage, megakaryocyte [CFU-GEMM]), Copeptin, AVP, AVPR1B agonist(s), EPO, or a combination thereof, are added to the media in a dosage from 10 ng to about 500 pg each in 100 pl NaCl solution, and colony-forming unit megakaryocytic (CFU-MK), and erythroids and white blood cell colonies in the different treatments are counted. For pre-treatment, CD34+ cells or LSK cells are left unstimulated or treated with copeptin, AVP, AVPR1B agonist(s), EPO, or a combination thereof, in a dosage from about 10 ng to about 500 pg in 100 mΐ NaCl solution, and the effect of treatment is determined after 30 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 12 hours, 24 hours and 48 hours. Colonies are counted using a STEMvision automated CFU colony counter (STEMCELL Technologies).

Differentiation Cultures

Undifferentiated CD34+ cells or LSK cells are treated with collagenase IV (1 mg/ml;

Invitrogen) and transferred to low attachment plates (Nunc) in liquid culture medium (Iscove’s modified Dulbecco’s medium - glutamax, Biochrom) containing human plasma in the presence of stem cell factor (SCF, 100 ng/ml), thrombopoietin (TPO, 100 ng/mL), FLT3 ligand (FL, 100 ng/mL), recombinant human bone morphogenetic protein 4 (BMP4; 10 ng/mL), recombinant human vascular endothelial growth factor (VEGF-A165; 5 ng/mL), interleukin-3 (IL-3; 5 ng/mL), interleukin-6 (IL-6; 5 ng/mL) (Peprotech) and erythropoietin (Epo; 3 U/mL) (Eprex). Copeptin,

A VP, AVPR1B agonist(s), EPO, or a combination thereof, are added to the media in a dosage fromlO ng to about 500 pg each in 100 pl NaCl solution. For pre-treatment, CD34+ cells or LSK cells are left unstimulated or treated with copeptin, AVP, AVPR1B agonist(s), EPO, or a combination thereof, in a dosage from about 10 ng to about 500 pg in 100 mΐ NaCl solution, prior to treatment with collagenase IV. The cells are cultured for 20 days at 37 °C in a humidified 5%

C0 ₂ atmosphere, and dissociated into a single-cell suspension by incubation with collagenase B (0.4 U/mL; Roche Diagnostics) for 30 min at 37°C and cell dissociation buffer (Invitrogen) for 10 min at 37°C. The cells are then plated at a density of 10 ⁶ cells/mL in liquid culture medium containing 10% human plasma, insulin 10 pg/mL and 3 U/mL heparin, in the presence of SCF (100 ng/mL), IL-3 (5 ng/mL) and EPO (3 U/ml). Erythrocyte, myeloid cell and platelet maturation is evaluated regularly based on cell morphology after staining and flow cytometry analysis.

Flow Cytometry analysis

Samples from the differentiation culture assays containing differentiated and

undifferentiated CD34+ cells or LSK cells are prepared in PBS-FBS (PBS containing 0.05% sodium azide, 1 mM EDTA, and 2% FBS), supplemented with 2% normal serum (Sigma- Aldrich), labeled with a combination of monoclonal antibodies and 7-aminoactinomycin D (7-AAD) for dead cell exclusion, and analyzed by flow cytometery. Intracellular staining is performed using FIX & PERM cell permeabilization reagents (Invitrogen). Samples are analyzed using a FACS Calibur flow cytometer (BD Biosciences) with CellQuest acquisition software (BD Biosciences). Control staining with appropriate isotype-matched control mAbs (BD Biosciences) is included to establish thresholds for positive staining.

Example 9

Role of copeptin and AVP, AVPR1B agonist(s), EPO, and combinations thereof on BM stromal cells

Bone marrow stromal cells are multipotent stem cells which form bone, cartilage and fat. Bone cells include osteoblasts (bone forming cells), osteoclast (bone removing cells) and osteocytes (bone maintaining cells); cartilage is made up of chondrocytes; and fat cells are adipocytes.

Therefore, to evaluate the role that copeptin, AVP, AVPR1B agonist(s), EPO, or a combination thereof, have in bone marrow (BM) function in vitro (separately as well as their synergistic effect), bone marrow stromal cells (BMSCs) are unstimulated or stimulated with different dosages of copeptin, AVP, AVPR1B agonist(s), EPO, or a combination thereof, at different times as described below, and their effect is determined by colony forming assay, differentiation culture assay and flow cytometry analysis.

BMSCs are freshly isolated from human or mice femurs and tibias by capping the bones.

The bone marrow is flushed out using a syringe filled with PBS, filtered through a 70- pm nylon mesh, centrifuged, and resuspended in DMEM growth medium supplemented with 20% FBS and penicillin/streptomycin. The cells are plated in 75-cm ² flasks, and kept at 37°C in a humidified atmosphere containing 95% air and 5% C0 ₂ . About 24 h after plating, supernatant containing non adherent cells is removed, and fresh medium is added. High density cultures are passaged on day 9 and well-separated colonies are selected for passage between day 13 to 17 (with media replacement at days 7 and 14) using cloning cylinders. BMSCs are harvested when primary cultures become ~70%-80% confluent.

Colony Forming Assay

Unstimulated and stimulated BMSCs are cultured in a semi-solid methylcellulose matrix supplemented with fibroblast progenitor cells (CFU-F), adipocyte progenitor cells (CFU-ADIPO), osteoblast progenitor cells (CFU-Ob) and chondrocyte progenitor cells (CFU-Ch). Copeptin, AVP. AVPR1B agonist(s), EPO, or a combination thereof, are added to the media in a dosage fromlO ng to about 500 pg in 100 pl NaCl solution, and osteoblast, adipocyte and chondrocyte colonies in the different treatments are counted. For pre-treatment, the BMSCs are left unstimulated or treated with copeptin, AVP, AVPR1B agonist(s), EPO, or a combination thereof, in a dosage from about 10 ng to about 500 mg in 100 mΐ NaCl solution, and the effect of treatment is determined after 30 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 12 hours, 24 hours and 48 hours. The differentiated cells are stained and the number of positive colonies is determined. Positive staining is defined as a colony that contains at least 20 positive cells (CFU-F, CFU-Ad) or 1 bone or cartilage nodule (CFU-Ob, CFU-Ch, respectively).

Differentiation Cultures

Undifferentiated BMSCs are maintained in osteoinductive or adipoinductive liquid medium at a density of 50,000 cells/well in 24-well plates. The medium is changed every three days.

Copeptin, A VP, AVPR1B agonist(s), EPO, or a combination thereof, are added to the media in a dosage fromlO ng to about 500 pg in 100 mΐ NaCl solution. For pre-treatment, BMSCs are left unstimulated or treated with copeptin, AVP, AVPR1B agonist(s), EPO, or a combination thereof, in a dosage from about 10 ng to about 500 pg in 100 pl NaCl solution, prior to transfer to

osteoinductive or adipoinductive liquid medium. At the end of a 3-week induction period, differentiation is assessed by alizarin red staining (for example by examining osteoblasts, adipocytes, and chondrocytes).

Flow Cytometry Analysis

Samples from the differentiation culture assays containing differentiated and

undifferentiated BMSCs are washed in PBS twice, stained with the respective antibodies directly conjugated to fluorescein isothiocyanate (FITC) at 4°C for 30 min, and resuspended in 300 pl PBS. Samples are analyzed using a FACS Calibur flow cytometer (BD Biosciences) with CellQuest acquisition software (BD Biosciences). Control staining with appropriate isotype- matched control mAbs (BD Biosciences) is included to establish thresholds for positive staining.

Example 10

Effect of copeptin on hematopoietic, T and B cell differentiation in vivo

Mice are kept at controlled temperature (22-24°C) and light/dark conditions for at least seven days and then injected subcutaneously with copeptin at different doses, such as between 10 ng/kg and 500 pg/kg, in 100 mΐ of 0.9% NaCl solution, twice daily throughout the experiment. Control mice are injected with placebo NaCl solutions. Bone marrow, spleen and blood samples are collected from the mice at different time points starting 12 hour after injection and up to seven days after injection. The samples are then analyzed by flow cytometry, colony forming assays and differentiation cultures to determine the effect of copeptin injection on red blood cell, white blood cell, T cell and B cell formation and functionality, as described in the examples above.

Example 11

Effect of copeptin on HSC, T cell and B cell transplantation

CD34+ cells or LSK cells are obtained from donor CD45.2+ and competitor CD45.1+ mice as described in the examples above. The cells are cultured and left unstimulated or treated with copeptin at a dosage from about 10 ng to about 500 pg in 100 mΐ NaCl solution. Recipient mice are kept at controlled temperature (22-24°C) and light/dark conditions for at least seven days and then irradiated with a red heat lump to dilate blood vessels. For competitive transplants, the irradiated mice are then injected subcutaneously with the un stimulated or copeptin-treated cells derived from both the donor and competitor mice in a 1: 1 ratio at different doses, such as between 10 ng/kg and 500 pg/kg, in 100 mΐ of 0.9% NaCl solution, twice daily throughout the experiment. For non competitive transplants, the irradiated mice are injected subcutaneously with the unstimulated or copeptin-treated cells derived from either the donor or competitor mice at different doses, such as between 10 ng/kg and 500 pg/kg, in 100 mΐ of 0.9% NaCl solution, twice daily throughout the experiment. Control mice are injected with placebo NaCl solutions. These assays allow comparison of stem cells derived from mice of different genotypes and determination of genotype effect on hematopoietic, T and B cell formation. Bone marrow, spleen and peripheral blood samples are collected from the mice at different time points starting one month after transplantation and up to six months after transplantation. The samples are then analyzed by flow cytometry, colony forming assays and differentiation cultures to determine the effect of transplantation on red blood cell, white blood cell, T cell and B cell formation and functionality, as described in the examples above.

Example 12

Effect of copeptin on stromal cell transplantation in vitro

Human and mice BMSCs are isolated and cultured as described in Example 9. Copeptin is added to the culture media in a dosage fromlO ng to about 500 pg in 100 pl NaCl solution. Control cells are treated with NaCl solution. After several passages to generate a sufficient number of cells, the cultured cells are seeded on a collagen or ceramic scaffolds, and then transplanted subcutaneously into immunocompromised (nude) mice. The transplants are harvested after 6 to 12 weeks, and the histology of the tissues formed is assessed for multipotency and to determine the effect of copeptin on hematopoiesis, T and B cell formation and bone, cartilage and adipocyte development.

Example 13

Identification of compounds that bind the copeptin receptor

The copeptin receptor is isolated and characterized by ligand affinity chromatography. The receptor is then purified and sequenced, and different natural and synthetic compounds are assayed by ligand affinity chromatography for their ability to bind the receptor and mimic copeptin activity.

Example 14

In vivo effect of copeptin, vasopressin and/or AVPPR1B agonist(s) on bone metabolism

A subject with central diabetes insipidus is administered copeptin, for example in combination with vasopressin (A VP) and/or AVPR1B agonist(s), at variable doses of 5-20 pg/day. In some examples, the copeptin, AVP and/or AVPR1B agonist(s) is linked to an immunoglobulin Fc domain. Bone metabolism is assessed by determining serum calcium, phosphorus and creatinine, circulating alkaline phosphatase and intact PTH. Bone mass density (BMD) is assessed by measuring bone density in the lumbar spine and in the femoral neck of the subject. The subject is considered osteopenic if the T score is between -1 and -2.5, and osteoporotic if the T score is lower than -2.5. At the end of treatment, the subject presents calcium, phosphorus, creatinine, alkaline phosphatase and PTH in the normal range, and significantly increased BMD values.

In view of the many possible embodiments to which the principles of our invention may be applied, it should be recognized that illustrated embodiments are only examples of the invention and should not be considered a limitation on the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.