WO2017165515A1 - Stimulation of bone growth using apolipoprotein e - Google Patents

Abstract

A method of stimulating bone growth in a subject is described that includes administering a therapeutically effective amount of Apolipoprotein E, an analog or homolog thereof, or an Apo E mimetic peptide, to the subject. Apolipoprotein E or its analogs, homologs, or mimetic peptides can be administered to a subject who has been diagnosed as having a bone growth disease or disorder such as osteogenesis imperfect.

Description

STIMULATION OF BONE GROWTH USING APOLIPOPROTEIN E

CONTINUING APPLICATION DATA

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 62/312,281, filed March 23, 2016, the disclosure of which is incorporated by reference herein.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on March 15, 2017, is named Sequence Listing for NCH-025160 WO ORD WorkFile and is 2,827 bytes in size.

BACKGROUND

[0002] Osteogenesis imperfecta (OI) is a hereditary disorder caused by mutations in type I collagen, which is the main component of bone. Besides bone weakness, short stature is another major symptom of OI, which can cause immaturity of the respiratory system by reducing the size of the chest cavity. Currently, there is no cure for OI and bisphosphonates are the standard treatment to strengthen bones. Palomo et al., J. of Bone and Minr Res, 30, 2150-2157 (2015). However, there is no such treatment for bone growth. Even growth hormone has demonstrated limited effects on bone growth in OI. Marini et al., J. Bone Miner Res., 18, 237-243 (2003). Thus, given that respiratory failure commonly causes death in OI patients, there is a critical need for developing effective treatments to reliably stimulate bone growth in this disorder. McAllion et al., J. of Clinical Pathology, 49, 627-630 (1996).

[0003] Mesenchymal Stem Cell (MSC) therapy has been applied to various diseases; however, there are several issues that require improvement before it can be applied to diseases such as OI. First, the therapeutic effects are not always consistent between MSCs from different donors. Zhukareva et al., Cytokine, 50, 317-321 (2010). Second, in contrast to culture-expanded MSCs, freshly-thawed, cryo-preserved MSCs fail to achieve therapeutic effects, limiting MSC therapy to patients at hospitals with Good Manufacturing Practice (GMP) facilities. Francis et al., Cytotherapy, 14, 147-152 (2012); Moll et al. Stem Cells, 32, 2430-2442 (2014). Finally, infusion of culture-expanded MSCs still has several unresolved safety concerns that could be problematic, such as malignant transformation, support of tumor growth, venous thrombosis, and pulmonary embolism. Miura et al., Stem Cells, 24, 1095-1103 (2006); Klopp et at., Stem Cells, 29, 11-19 (2011); Barbash et al, Circulation 108, 863-868 (2003). Therefore, the development of treatments that provide equivalent therapeutic effects without culture-expanded MSCs would be a major advance in the field.

[0004] Towards this, the inventors' trials of MSC therapy for clinical treatment of OI demonstrated growth acceleration in OI patients after systemic infusions of MSC (average 3.5 fold increase of growth rate). Otsuru et al, Blood, 120, 1933-1941 (2012). Despite the striking growth, the therapeutic effects lasted only 6 months. Thus, to provide steady and enduring growth for patients this transient growth must be overcome. Since the mechanism of bone growth after MSC infusion involves multiple factors and steps, there is a critical need to identify the exact mechanism of how MSC infusion induces bone growth. Horowitz et al., Proc Natl Acad Sci, 99, 8932-8937 (2002).

SUMMARY OF THE INVENTION

[0005] Bone growth occurs in a bone's growth plate. Both OI patients and mice have an elongated growth plate, especially in the hypertrophic zone (HZ), where fewer proliferating chondrocytes are seen. Evans et al., Genetic Disorders 32, 268-272 (2003). Using a mouse model of OI, the inventors found that infused MSCs induce bone growth by stimulating the release of a soluble factor(s) (designated as chondrocyte factor(s)) from as yet undetermined tissue(s) into the serum, which enhances chondrocyte proliferation in the growth plate.

[0006] Further investigation successfully isolated apo lipoprotein E (ApoE) as the primary candidate for the factor. In support of this, the inventors verified that the ApoE levels in the serum were significantly elevated after MSC infusion. Moreover, the inventors found that ApoE knockout mice have a markedly reduced body size. Consistent with this, OI mice have significantly reduced levels of ApoE in their bones. Therefore, the inventors hypothesize that ApoE stimulates bones growth in OI by stimulating chondrocyte proliferation and differentiation in bone growth plates as a mediator of MSC-induced therapeutic effects. Previous work has shown that Apolipoprotein E inhibits osteoclast differentiation. Kim et al., Exp Cell Res., 319(4), 436-46 (2013). [0007] Recombinant ApoE has been shown to stimulate proliferation and differentiation of in vitro cultured chondrocytes and bone growth in ex vivo bone organ cultures. Additionally, systemic administration of recombinant ApoE has been shown to stimulate bone growth in OI model mice. Therefore, in vivo application of recombinant ApoE promotes chondrocyte proliferation similarly to MSC infusions, but without the potential complications associated with those infusions. Thus, ApoE can be safely and repeatedly implemented during the growth period to initiate and maintain bone growth in OI patients and overcome the problem of transient growth observed in MSC-infused patients.

BRIEF DESCRIPTION OF THE FIGURES

[0008] The present invention may be more readily understood by reference to the following figures, wherein:

[0009] Figure 1 provides a scheme showing a novel mechanism of bone growth after MSC infusion. Infused MSCs induce the produce of chondrocyte factor that acts on the growth plate chondrocytes.

[0010] Figures 2A-2D provide a schematic diagram and graphs showing the results of a chondrocyte proliferation assay. (A) Schematic diagram of the chondrocyte proliferation assay to test the sera from MSC- or PBS-infused mice. (B, C) MSC infusion promoted the induction of chondrocyte factor both in wild type and OI model mice. (D) Ex vivo bone organ culture using the positive and negative sera. (Positive serum: serum from MSC infused mice, Negative serum: serum from PBS infused mice).

[0011] Figures 3A-3E provides graphs relating to the characterization of the chondrocyte factor(s). Heat (HI, 95 °C for 10 min or 56 °C for 20 min) treatment (A) and proteinase K (ProtK) treatment (B) inactivated the chondrocyte factor. (C) When the >300 kDa fraction was removed, the positive serum lost the activity whereas the negative did not. (D) When the >300 kDa fraction was switched between the positive and negative serum, the positive serum lost activity, while the negative gained it. (E) Positive and negative sera were fractionated with FPLC. The first peak was unique to the positive serum (red arrow). Molecular weight standard is show in blue. [0012] Figures 4A & 4B provide graphs showing the blockade of LDLR and Scrabl. (A) Chondrocyte proliferation was suppressed in the presence of neutralizing antibodies for LDLR and Scrabl . (B) Chondrocytes isolated from LDLR knockout (KO) mice (LDLR -/-) and Scrabl KO (Scrabl -/-) showed significantly less proliferation compared to wild type chondrocytes when they were cultured with the positive serum.

[0013] Figures 5A-5D provide graphs showing the effect of ApoE on chondrocyte proliferation. (A) Serum level of ApoE was significantly higher in the positive serum. (B) Addition of ApoE into the negative serum stimulated chondrocyte proliferation. Adding 400 ng/ml of ApoE into the negative serum improved chondrocyte proliferation to a similar degree as the positive serum. (C) Addition of ApoE into the negative serum also stimulated bone growth in the ex vivo bone organ culture. (D) ApoE level in femora was significantly reduced in OI model mice.

[0014] Figures 6A-6D provide graphs and images of growth plate analyses. (A) Femur length (black solid: wild type, blue dash: OI). (B) H&E staining of the growth plate. (1) between two blue lines: growth plate height (C). (2) between dash line and lower blue line: hypertrophic zone height (D).

[0015] Figure 7 provides graphs showing gene expressions in chondrocyte pellet culture. Expression of osteoblast related genes (Runx2, Osteopontin (OPN), Collagen I (Coll), Alkalinephosphatase (ALP), osterix (Osx), bonesialoprotein (BSP) and bone gla protein (BGLAP) was analyzed in the chondrocyte pellet after 3 weeks culture with/without ApoE (black: without, gray: with ApoE). The expression is shown relative to that in wild type pellet after 1 week culture. N.S.: not significant.

[0016] Figures 8A-8C provides the A) nucleotide sequence for the gene encoding Apolipoprotein E (SEQ ID NO: 1), B) the amino acid sequence for the Apolipoprotein E protein (SEQ ID NO: 2) (the first 18 amino acids are signal peptides), and the amino acid sequence for recombinant Apolipoprotein E lacking the signal peptide sequence (SEQ ID NO: 3).

[0017] Figures 9A and 9B provide a scheme and graph showing A) the steps involved in a loss-of-function study and B) the relative proliferation of chondrocytes in response to serum from wild-type (WT), Osteogenesis imperfect (OI) and ApoE knockout mice infused with either MSC or PBS.

[0018] Figures 10A and 10B provide a timeline and graph showing A) the schedule of MSC infusion in OI mice and B) the body weight, femur length, and tibia length of OI mice pre- treatment and post-treatment, where some OI mice received only phosphate buffered saline (PBS) while other OI mice received MSC cells.

[0019] Figures 11A and 1 IB provide a timeline and graph showing A) the schedule of MSC infusion in OI mice lacking the ApoE gene, and B) the body weight, femur length, and tibia length of OI mice lacking the ApoE gene pre-treatment and post-treatment, where some OI mice received only phosphate buffered saline (PBS) while other OI mice received MSC cells.

[0020] Figures 12A and 12B provide a timeline and graph showing A) times of implantation of an osmotic pump containing either PBS or 170 μg of recombinant ApoE in OI mice, and B) the body weight, femur length, and tibia length of OI mice prc-treatment and post- treatment, where some OI mice received only PBS while other OI mice received recombinant ApoE.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention relates to a method of stimulating bone growth in a subject, comprising administering a therapeutically effective amount of Apolipoprotein E (Apo E), an analog or homolog thereof, or an Apo E mimetic peptide, to the subject.

Definitions

[0022] As used herein, the terms "treatment", "treating", and the like, refer to obtaining a desired pharmacologic or physiologic effect. The effect may be therapeutic in terms of a partial or complete cure for a disease or an adverse effect attributable to the disease. "Treatment", as used herein, covers any treatment of a disease in a mammal, particularly in a human, and can include inhibiting the disease or condition, i.e., arresting its development; and relieving the disease, i.e., causing regression of the disease. [0023] Prevention, as used herein, refers to treatment of a subject identified as being at risk of being afflicted with a condition or disease such as osteogenesis imperfecta, including avoidance of development of a bone disease or disorder, or a decrease of one or more symptoms of the bone disease or disorder should a bone disease or disorder develop nonetheless. Accordingly, Apolipoprotein E, or an analog or homolog thereof, or an Apo E mimetic peptide, can be administered prophylactically to a subject who has not been diagnosed as having a bone disease or disorder.

[0024] "Pharmaceutically acceptable" as used herein means that the compound or composition is suitable for administration to a subject for the methods described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.

[0025] The terms "therapeutically effective" and "pharmacologically effective" are intended to qualify the amount of an agent which will achieve the goal of improvement in disease severity and the frequency of incidence. The effectiveness of treatment may be measured by evaluating a reduction in psychotic symptoms in a subject in response to the administration of antipsychotic agents.

[0026] As used herein, the term "diagnosis" can encompass determining the likelihood that a subject will develop a disease, or the existence or nature of disease in a subject. The term diagnosis, as used herein also encompasses determining the severity and probable outcome of disease or episode of disease or prospect of recovery, which is generally referred to as prognosis). "Diagnosis" can also encompass diagnosis in the context of rational therapy, in which the diagnosis guides therapy, including initial selection of therapy, modification of therapy (e.g., adjustment of dose or dosage regimen), and the like.

[0027] A "subject." as used herein, can be any animal, and may also be referred to as the patient. Preferably the subject is a vertebrate animal, and more preferably the subject is a mammal, such as a domesticated farm animal (e.g., cow, horse, pig) or pet (e.g., dog, cat). In some embodiments, the subject is a human.

[0028] The term "polynucleotide" or "nucleic acid molecule" refers to a polymeric form of nucleotides of at least 10 bases in length. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native inter- nucleoside bonds, or both. The nucleic acid can be in any topological conformation. For instance, the nucleic acid can be single-stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hair-pinned, circular, or in a padlocked conformation.

[0029] The terms "polypeptide" and "peptide" are used interchangeably herein to refer to a polymer of amino acids. These terms do not connote a specific length of a polymer of amino acids. Thus, for example, the terms oligopeptide, protein, and enzyme are included within the definition of polypeptide or peptide, whether produced using recombinant techniques, chemical or enzymatic synthesis, or naturally occurring. This term also includes polypeptides that have been modified or derivatized, such as by glycosylarion, acetylation, phosphorylation, and the like.

[0030] "Amino acid" is used herein to refer to a chemical compound with the general formula: NH₂-CRH-COOH, where R, the side chain, is H or an organic group. Where R is organic, R can vary and is either polar or nonpolar (i.e., hydrophobic). The amino acids of this invention can be naturally occurring or synthetic (often referred to as nonproteinogenic).

[0031] The following abbreviations are used throughout the application: A Ala = Alanine, T = Thr = Threonine, V = Val = Valine, C = Cys = Cysteine, L = Leu = Leucine, Y = Tyr = Tyrosine, I = He = Isoleucine, N = Asn = Asparagine, P Pro = Proline, Q = Gin - Glutamine, F = Phe = Phenylalanine, D = Asp = Aspartic Acid, W = Trp = Tryptophan, E Glu = Glutamic Acid, M = Met = Methionine, K = Lys = Lysine, G = Gly = Glycine, R = Arg Arginine, S = Ser - Serine, H - His - Histidine.

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0033] As used herein and in the appended claims, the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide" also includes a plurality of such peptides. [0034] In one aspect, the present invention provides a method of stimulating bone growth in a subject comprising administering a therapeutically effective amount of Apolipoprotein E (Apo E), an analog or homolog thereof, or an Apo E mimetic peptide, to the subject. In some embodiments, Apo E or an analog or homolog thereof are administered, while in other embodiments, an Apo E mimetic peptide is administered.

Apolipoprotein E

[0035] Apolipoprotein E is a class of apolipoprotein found in the chylomicron and Intermediate-density lipoprotein (IDLs) that is essential for the normal catabolism of triglyceride-rich lipoprotein constituents. Human Apo E is 299 amino acids long and contains multiple amphipathic a-helices. The gene encoding human Apo E (APOE), is mapped to chromosome 19 in a cluster with Apolipoprotein CI and the Apolipoprotein CI. The APOE gene consists of four exons and three introns, totaling 3597 base pairs. The amino acid sequence (SEQ ID NO: 2) for the protein (including signal peptides), and the nucleic acid sequence (SEQ ED NO: 1) of cDNA encoding the protein, arc shown in Figure 8. The signal peptide is included in natural forms of Apo E, but is not required for activity and is not included in recombinant forms of Apo E (SEQ ID NO: 3).

[0036] Apo E is polymorphic with three major alleles: ApoE2 (cysl l2, cysl 58), ApoE3 (cysl l2, argl58), and ApoE4 (argl 12, argl 58). Ghebranious et al_^ Nucleic Acids Research 33 (17): el 49 (2005). Apo E3 is the most common form, having an allele frequency of approximately 79 percent, and is considered the "neutral" Apo E genotype. In some embodiments, the ApoE3 allele isoform of Apo E is administered.

[0037] By "homologs" what is meant is peptides closely corresponding to the sequences identified for Apolipoprotein E, including sequences from other mammalian species that are substantially homologous at the overall protein {i.e., mature protein) level to human Apolipoprotein E (see Figure 8), so long as such homologous peptides retain their respective known activities. Various levels of homology, from 55% to 99%, are described herein. For example, preferred levels of homology include 75%, 85%, 90%, and 95%.

[0038] By "analogs" is meant peptides which differ by one or more amino acid alterations, which alterations, e.g., substitutions, additions or deletions of amino acid residues, do not abolish the properties of the relevant peptides, such as their ability to inhibit angiogenesis. An analog may comprise a peptide having a substantially identical amino acid sequence to a peptide provided herein and in which one or more amino acid residues have been conservatively or non-conservatively substituted. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another. Likewise, the present invention contemplates the substitution of one polar (hydrophilic) residue such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine or histidine for another or the substitution of one acidic residue such as aspartic acid or glutamic acid for another is also contemplated. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, leucine, alanine, methionine for polar (hydrophilic) residues such as cysteine, glutamine, glutamic acid, lysine and/or a polar residue for a non-polar residue.

[0039] The phrase "conservative substitution" also includes the use of chemically derivatized residues in place of a non-derivatized residues as long as the peptide retains the requisite activity (e.g., anti-angiogenic activity). Analogs also include the presence of additional amino acids or the deletion of one or more amino acids which do not affect Apo lipoprotein E mediated biological activity. For example, analogs of the subject peptides can contain an N- or C-terminal cysteine, by which, if desired, the peptide may be covalently attached to a carrier protein, e.g., albumin. Such attachment can decrease clearing of the peptide from the blood and also decrease the rate of proteolysis of the peptides. In addition, for purposes of the present invention, peptides containing D-amino acids in place of L-amino acids are also included in the term "conservative substitution." The presence of such D-isomers can help minimize proteolysis and clearing of the peptide.

[0040] Ordinarily, the conservative substitution variants, analogs, and derivatives of the peptides, will have an amino acid sequence identity to the disclosed sequences of at least about 55%, at least about 65%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% to 99%. Identity or homology with respect to such sequences is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the known peptides, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. N-terminal, C- terminal or internal extensions, deletions, or insertions into the peptide sequence shall not be construed as affecting homology.

[0041] In other embodiments, the method comprises administering an Apo E mimetic peptide to the subject. "Peptide mimetics" are structures which serve as substitutes for peptides in interactions between molecules (See Morgan et al., Ann. Reports Med. Chem. 24:243-252 (1989) for a review). Peptide mimetics include synthetic structures which may or may not contain amino acids and/or peptide bonds but retain the structural and functional features of a peptide, or enhancer or inhibitor of the invention. Peptide mimetics also include peptoids, oligopeptoids (Simon et al., Proc. Natl. Acad, Sci USA 89:9367 (1972)); and peptide libraries containing peptides of a designed length representing all possible sequences of amino acids corresponding to a peptide of the invention. In some embodiments, mimetic peptides are versions of Apo E having a substantially decreased amino acid length, yet retain the biological activity of Apo E. For example, Apo E mimetic peptides can have a length from 20 to 50 amino acids, or in some embodiments from 25 to 35 amino acids. Examples of Apo E mimetic peptides include AEM-28 and its analogs. See U.S. Patent No. 8,084,423 and U.S. Patent No. 8,557,767.

Treating or Preventing Bone Disease or Disorder

[0042] The present invention provides a method of stimulating bone growth in a subject. It will be understood that stimulating bone growth in a subject means elevating the level of osteogenesis and/or bone mass above that which exists prior to treatment in accordance with the present invention. For example, a change in the level can readily be determined by comparing levels in a subject before and after treatment. The level of osteogenesis and/or bone mass before and/or after treatment may be determined from a series of measurements taken over different timepoints to provide a standard range. The level of osteogenesis and/or bone mass before and/or after treatment may be measured in multiple individuals to provide a standard range representative of a given population. In certain embodiments, the level of osteogenesis and/or bone mass may be increased in a subject treated by the methods of the present invention by at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%, compared to the level of osteogenesis and/or bone mass prior to treatment. [0043] In some embodiments, subjects in which bone growth is stimulated may be suffering from a bone growth disease or disorder, or suspected to be suffering from a bone growth disease or disorder. Alternatively, subjects treated may not be suffering from a bone growth disease or disorder, but may be susceptible to suffering from a bone growth disease or disorder. For example, a subject susceptible to suffering from a bone growth disease or disorder can be a subject that has been diagnosed as having an increased risk of developing a bone growth disease or disorder. A determination of whether a given subject is suffering from a given bone growth disease or is susceptible to a given bone growth disease can be made by those of skilled in the art based on clinical symptoms and/or other standard diagnostic tests which may vary depending on the particular disease in question.

[0044] As used herein, "bone growth disease" or "bone growth disorder" refers to a disease or condition associated with abnormality of the bone that can be treated by increasing bone mass and/or bone growth. A wide variety of bone growth disease and disorders are known to those skilled in the art. Examples of bone growth diseases and disorders include, but are not limited to: Achondrogenesis; Achondroplasia; Acrodysostosis; Acromesomelic Dysplasia (Acromesomelic Dysplasia Maroteaux Type, AMDM); Atelosteogenesis; Campomelic Dysplasia; Cartilage Hair Hypoplasia (CHH) (Metaphyseal Chondrodysplasia, McKusick type); Chondrodysplasia Punctata; Cleidocranial Dysostosis; Conradi-Hunermann Syndrome; Cornelia de Lange; Cranioectodermal dysplasia; Desbuquois syndrome; Diastrophic Dysplasia; Dyggve-Melchior-Clausen; Dyssegmental Dysplasia; Ellis van Creveld Syndrome (Chondroectodermal Dysplasia, EVC); Growth Hormone Deficiency; Hallerman-Streiff Syndrome; Hunter Syndrome (MPS II); Hurler-Scheie Syndrome (MPS I); Hypochondrogenesis; Hypochondroplasia; Hypophosphatasia; Hypophosphatemia; Hypopituitary; Hypothyroidism; Jarcho-Levin Syndrome (Spondylothoracic Dysplasia, Spondylocostal); Jeune Syndrome (Asphyxiating Thoracic Dysplasia; Asphyxiating Thoracic Dystrophy); Kniest Dysplasia; Laron Dwarfism; Larsen Syndrome; Leri- Weill Dyschondrosteosis (Mesomelic Dwarfism, Madelung Deformity); Lethal Skeletal Dysplasias; Maroteaux-Lamy (MPS VT) (MPS VI); Mesomelic Dysplasia; Metaphyseal Chondrodysplasia- Jan sen Type; Metaphyseal Dysplasia-Schmid Type; Metatropic Dysplasia; Morquio Syndrome (MPS IV); Mucopolysaccharidoses; Multiple Epiphyseal Dysplasia (MED); Osteogenesis Imperfecta (OI); Pituitary Dwarfism; Precocious Puberty; Primordial Dwarfism (Microcephalic Osteodysplastic Primordial Dwarfism, MOPD); Pseudoachondroplasia; Rhizomelic Chondrodysplasia Punctata; Rickets; Robinow dwarfism/syndrome; Russell-Silver Syndrome; SADDAN: Severe Achondroplasia with Acanthosis Nigricans and Developmental Delay; Schmike Immuneosseous Dysplasia; Seckel Syndrome; Short Rib Polydactyly; Shwachman -Diamond Syndrome; Spondyloepimetaphyseal Dysplasia-Strudwick (SEMD); Spondyloepimetaphyseal dysplasias; Spondyloepiphyseal Dysplasia; Spondyloepiphyseal Dysplasia Congenita (SED-Congenita, SEDC); Spondyloepiphyseal Dysplasia Tarda (SED-Tarda, SEDT, SED-L); Spondylom etaphyseal Dyspl asia-Corner Fracture Type (SMD, SMD-Corner Fracture Type, SMD-Sutcliffe Type); Spondylometaphyseal Dysplasia-Kozlowski (SMD-Kozlowski, SMDK); Thanatophoric Dysplasia; Trichorhinophalangeal Syndrome (Langer-Giedion syndrome); Turner Syndrome; Type II Collagenopathies.

[0045] hi some embodiments, the subject has been diagnosed as having bone growth disease or disorder selected from the group consisting of osteogenesis imperfecta, disorders caused by increased osteoclastogenesis or bone loss associated with inflammatory conditions, infection, genetic and age-related bone disorders such as osteoporosis, osteopenia, Paget's disease, metastatic bone cancer, myeloma bone disease, bone fracture healing, and bone graft repair.

[0046] In some embodiments, the subject has been diagnosed as having osteogenesis imperfecta. Osteogenesis imperfect (OI), also known as brittle bone disease or Lobstein syndrome, is a congenital bone disorder characterized by brittle bones that are prone to fracture. People with OI are born with defective connective tissue, or without the ability to make it, usually because of a deficiency of type I collagen. Symptoms that can be used to diagnose osteogenesis imperfect include shorter height, neurological features including communicating hydrocephalus, basilar invagination, and seizures, blue sclerae, and hearing loss. Osteogenesis imperfect can be classified into 8 different types, based on the severity of the condition. A skin biopsy can be performed to determine the structure and quantity of type I collagen. DNA testing can be carried out to confirm the diagnosis.

[0047] The efficacy of methods for preventing or treating diseases provided herein may be determined using any suitable technique for quantifying the level of osteogenesis. Commercial kits for quantifying the level of osteogenesis are available (e.g. Millipore osteogenesis quantitation kit, Cat # ECM815). Additionally or alternatively, the level of osteogenesis can be determined by quantifying mRNA levels of genes associated with osteogenesis (e.g. RUNX2, OCN and OPN) using techniques such as RT-PCR, and/or other methods such as alkaline phosphatase assays, alizarin red-S and von Kossa staining, and the like.

[0048] Bone mass can be useful to determine if a subject is suffering from a bone growth disease or disorder, or to evaluate treatment of a bone growth disease or disorder. Bone mass may be measured in mammalian subjects (e.g. humans) using standard techniques (e.g. dual energy X-ray absorptiometry (DXA)). In particular, DXA may be used for diagnosis, prognosis (e.g. fracture prediction), monitoring the progression of a bone disease, and/or assessing responses to treatment. Categorization of subjects into diseased and non-diseased states based on bone mass (i.e. bone material density) can be made on the basis of standard classification systems including those published by the World Health Organization (see, for example, World Health Organization Technical Report Series 921 (2003), Prevention and Management of Osteoporosis). For example, a diagnosis of osteoporosis may be based on BMD that is two standard deviations or more below a young adult reference mean.

[0049] Bone disease or disorders characterized by a loss of bone mass also encompass abnormalities in the strength and structures of the bones. Examples include, but are not limited to, decreased bone mass, change in bone density, bone softness, tumors on bones and abnormal bone architecture.

[0050] Additionally or alternatively, a diagnosis of a given bone disease can be made by .> physician, nurse, or veterinarian, depending on the subject under consideration.

[0051] In some embodiments, the method of stimulating bone growth also includes administration of one or more additional bone growth stimulating agents. The term "bone growth-stimulating agent" is intended to include any material that stimulates and encourages the development and functional maintenance of bone, and mature osteoclasts in bone. Examples of bone growth-stimulating agents include, without limitation: growth factors; cytokines and the like, such as members of the Transforming Growth Factor-beta (TGF-β) protein superfamily including any of the Bone Morphogenic Proteins (BMP) and members of Glycosylphosphatidylinositol-anchored (GPI-anchored) signaling proteins including members of the Repulsive Guidance Molecule (RGM) protein family; other growth regulatory proteins, and bisphosphonates. [0052] Because patients with a bone disease or disorder have heterogeneous clinical manifestations and varying clinical outcomes, the treatment given to a subject may vary, depending on their prognosis. The skilled clinician will be able to readily determine without undue experimentation specific secondary agents, types of therapy that can be effectively used to treat an individual subject with a bone disease or disorder.

Administration of Apo lipoprotein E

[0053] The present invention also contemplates pharmaceutical formulations for administration to a subject (e.g., a human), which comprise as the active agent Apolipoprotein E and analogs and homologs thereof described herein. In such pharmaceutical and medicament formulations, the active agent is preferably utilized together with one or more pharmaceutically acceptable carrier(s) and optionally any other therapeutic ingredients. The carrier(s) must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not unduly deleterious to the recipient thereof The active agent is provided in an amount effective to achieve the desired pharmacological effect, as described above, and in a quantity appropriate to achieve the desired daily dose.

[0054] Typically, the Apolipoprotein E will be suspended in a sterile saline solution for therapeutic uses. The pharmaceutical compositions may alternatively be formulated to control release of active ingredient (Apolipoprotein E) or to prolong its presence in a patient's system. Numerous suitable drug delivery systems are known and include, e.g., implantable drug release systems, hydrogels, hydroxym ethyl cellulose, microcapsules, liposomes, microemulsions, microspheres, and the like. Controlled release preparations can be prepared through the use of polymers to complex or adsorb the molecule according to the present invention. For example, biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of a polyanhydride copolymer of a stearic acid dimer and sebaric acid. The rale of release of the molecule according to the present invention, i.e., of an antibody or antibody fragment, from such a matrix depends upon the molecular weight of the molecule, the amount of the molecule within the matrix, and the size of dispersed particles.

[0055] The pharmaceutical composition of this invention may be administered by any suitable means, such as orally, topically, intranasally, subcutaneously, intramuscularly, intravenously, intra-arterially, intraarticulary, intralesionally or parenterally. Ordinarily, intravenous (i.v.), intraarticular, topical or parenteral administration will be preferred. When administered, the peptide compositions may be combined with other ingredients, such as carriers and/or adjuvants. The peptides may also be covalently attached to a protein carrier, such as albumin, so as to decrease metabolic clearance of the peptides.

[0056] It will be apparent to those of ordinary skill in the art that the therapeutically effective amount of Apolipoprotein E will depend, inter alia upon the administration schedule, the unit dose of molecule administered, whether the peptide is administered in combination with other therapeutic agents, the immune status and health of the patient, the therapeutic activity of the peptide administered and the judgment of the treating physician.

[0057] Although an appropriate dosage of a molecule of the invention varies depending on the administration route, age, body weight, sex, or conditions of the subject, and should be determined by the physician in the end, in the case of oral administration, the daily dosage can generally be between about 0.01 mg to about 500 mg, preferably about 0.01 mg to about 50 mg, more preferably about 0.1 mg to about 10 mg, per kg body weight. In the case of parenteral administration, the daily dosage can generally be between about 0.001 mg to about 100 mg, preferably about 0.001 mg to about 10 mg, more preferably about 0.01 mg to about 1 mg, per kg body weight. The daily dosage can be administered, for example in regimens typical of 1-4 individual administration daily. Other preferred methods of administration include intraarticular administration of about 0.01 mg to about 100 mg per kg body weight. Dosage administered can also be measured by using a target serum concentration. For example, in some embodiments, a dosage can be administered to provide a serum concentration from 100 ng/mL. to 1000 ng/mL, from 200 ng/mL to 800 ng/mL, from 300 ng/mL to 500 ng/mL, or at least 400 ng/mL. Various considerations in arriving at an effective amount are described, e.g., in Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990.

[0058] The Apolipoprotein E can be dissolved, dispersed or admixed in an excipient that is pharmaceutically acceptable and compatible with the active ingredient as is well known. Suitable excipients are, for example, water, saline, phosphate buffered saline (PBS), dextrose, glycerol, ethanol, or the like and combinations thereof. Other suitable carriers are well known to those skilled in the art. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents.

[0059] In some embodiments, a single dose of Apo E is administered. However, in other embodiments, the Apo E or analog or homolog thereof is administered repeatedly or continuously over a significant period of time. This can be achieved either through repeated administration, or through use of a controlled-release formulation.

[0060] Examples have been included to more clearly describe a particular embodiment of the invention and its associated cost and operational advantages. However, there are a wide variety of other embodiments within the scope of the present invention, which should not be limited to the particular examples provided herein.

EXAMPLES

Example 1: Stimulation of Chondrocyte Proliferation in OI Model

[0061 ] To achieve the goal of developing treatments for osteogenesis imperfecta (OI) without differences observed between MSCs from different donors, freshly-thawed, cryo -preserved MSCs failing to achieve therapeutic results, and culture-expanded MSCs potentially causing malignant transformation, support tumor growth, or venous thrombosis and pulmonary embolism, the identification of how MSCs achieve therapeutic activity is the first critical step to developing novel, cell-free therapies. This investigation has been executed using the general model of Fig. I . A murine model of OI was infused with MSCs to determine how they promote bone growth by inducing the release of a soluble(s) chondrocyte factor(s) into the serum, which then stimulate the proliferation of chondrocytes in the growth plate.

[0062] in vitro chondrocyte proliferation assay (Bioassay): As previously shown, MSCs did not directly stimulate chondrocyte proliferation. Otsuru et al., Blood, 120, 1933-1941 (2012). Thus, to detect the chondrocyte factors) in the serum, the inventors developed an in vitro chondrocyte proliferation assay. Otsuru et al., Cytotherapy, 17, 262-270 (2015); Otsuru et al., Blood, 120, 1933-1941 (2012). In this assay, freshly isolated chondrocytes from murine neonates are cultured for 6 days in medium supplemented with serum (1%) from mice intravenously infused with MSCs or PBS (Fig. 2A). Proliferation is measured by the intensity of a fluorescent dye binding to DNA using a plate reader. They verified that the sera from MSC infused wild type mice (positive serum) stimulated approximately 30% more chondrocyte proliferation than the sera from PBS infused wild type mice (negative serum) (Fig. 2B). Similar stimulation of chondrocyte proliferation was observed in OI mice as well (Fig. 2C), suggesting that the release of the chondrocyte factor(s) following MSC infusion is not a phenomenon specific to OI. Ex vivo bone organ culture using femora and tibiae from day 4 neonates showed that the positive serum stimulated bone growth greater than the negative serum (Fig. 2D), indicating that chondrocyte proliferation enhanced by the positive serum correlated to bone growth.

Example 2: Characterization of the Chondrocyte Factor(s).

[0063] To characterize the chondrocyte factor(s) in the serum, the inventors treated the positive sera with heat and proteinase K, and then tested the treated sera in their bioassay (Figs. 3 A, 3B). These treated sera failed to stimulate chondrocyte proliferation, indicating factor(s) is a protein.

[0064] The inventors then serially fractionated the positive and negative sera, into 5 fractions (0-10 kDa, 10-50 kDa, 50-100 kDa, 100-300 kDa, and >300 kDa) using size exclusion spin columns. To determine which fraction has the factor(s) providing additional chondrocyte proliferation, they recombined all fractions "minus one" and tested these mixed fractions in their bioassay. Compared to the mixture of all fractions from each serum, mixtures lacking either the 0-10 kDa or 100-300 kDa showed significantly less chondrocyte proliferation, however this reduction was similarly seen in both fractionated positive and negative sera, suggesting that these fractions contain growth factors common to both positive and negative sera. However, when the >300 kDa fraction was omitted, chondrocyte proliferation was significantly reduced in the positive serum but not in the negative serum (Fig. 3C). These data suggested that the positive serum has the chondrocyte factor(s) in the >300 kDa fraction. Moreover, they made mixtures by switching a single fraction between the positive and negative sera and then examined these mixtures in their bioassay. When fractions between 0 to 300 kDa were switched, chondrocyte proliferation was unaffected suggesting that both positive and negative sera contain similar amounts of comparable growth factors and cytokines in these fractions. However, when the >300 kDa fraction was switched, the positive serum lost additional activity while the negative serum gained stimulatory activity in their bioassay, compared to the unswitched negative serum (Fig. 3D). These data, also suggest that the chondrocyte factor(s) is in the >300 kD fraction of the positive serum.

[0065] Consistent with these results, fast protein liquid chromatography (FPLC) analyses with the both positive and negative sera detected a unique peak in high molecular weight fractions (>300 kDa) of the positive serum (Fig. 3E). This peak was not observed in the negative serum. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of fractions in this peak identified 13 proteins specific to the positive serum and 18 proteins significantly more abundant in the positive serum. These 31 candidates for the chondrocyte factors) contained eight apolipoproteins and seven complements. None of these candidates except one had molecular weight higher than 300 kDa, suggesting that they form complexes in the >300 kDa fraction.

Example 3; Database Analysis of Chondrocyte Factor Candidates

[0066] Database search: In order to respond to chondrocyte factor(s) present in serum, chondrocytes must express receptors for the factor(s) or proteins that bind the factor(s) on their plasma membrane for further intracellular signal transduction. Thus, the inventors first searched for possible membrane proteins to which the 31 candidates can bind using "STRING" a database for known and predicted protein-protein interactions, and made a list of receptors/binding proteins for each candidate. RNA-sequencing was then performed using RNA purified from freshly isolated chondrocytes to obtain the gene expression profile. By cross referencing this gene expression profile with the STRING data, the inventors checked whether the receptors/membrane proteins on the lists are expressed by chondrocytes. These searches narrowed the list of 31 candidates down to 11 with 41 possible receptors/binding proteins shown in Table 1.

[0067] Table 1 provides a list of candidates for the chondrocyte factor(s) and its possible receptors/binding proteins. The eleven candidates identified from the database search are listed on the left column. The cell surface proteins on chondrocytes that the candidates can possibly bind to are listed on the top row. The binding of the candidates to the surface proteins is shown in black squares.

[0068] Table 1 : Candidates for the Chondrocyte Factor

Example 4: Blockade of LDLR and Scrabl

[0069] Since there were three apolipoproteins in the 11 candidates, the inventors first examined whether these apolipoproteins can contribute to chondrocyte proliferation. To block signals from apolipoproteins, they treated chondrocytes with neutralizing antibodies for Low Density Lipoprotein Receptor (LDLR) and scavenger receptor class B type I (Scarbl), which is known to be one of the high density lipoprotein receptors. These treated chondrocytes showed significantly less proliferation compared to those treated with the appropriate isotope control in the inventors' bioassay (Fig. 4A). To verify these findings, chondrocytes isolated from LDLR knockout mice and Scrabl knockout mice were used in the bioassay. Consistent with the results from the blockade with the neutralizing antibodies, chondrocytes from both knockout mice failed to respond to the chondrocyte factor(s) in the positive serum and the proliferation was significantly diminished (Fig. 4B). These results suggest that signals through LDLR and Scarbl are critical for the additional chondrocyte proliferation induced by the positive serum, and that the chondrocyte factors) is a protein that can bind to these receptors.

Example 5: Identification of the Primary Candidate for the Chondrocyte Factorfs).

[0070] According to Table 1 , apolipoprotein E (Apo E) is the only candidate protein which can bind to both LDLR and Scarbl, suggesting that ApoE is the primary candidate for the chondrocyte factor. To test this possibility, the inventors first examined whether MSC infusion induced the release of ApoE into the serum. ELISA for ApoE showed that the positive sera contained significantly higher levels of ApoE than the negative sera (Fig. 5A). They then checked whether chondrocyte proliferation could be stimulated with ApoE. When recombinant human ApoE3 was added into the medium in their bioassay, chondrocyte proliferation with the negative sera was significantly stimulated in a dose-dependent manner up to the equivalent level with the positive sera, suggesting that addition of ApoE into the negative serum compensated for the lack of ability to enhance chondrocyte proliferation (Fig. 5B). Moreover, ApoE stimulated bone growth in ex vivo bone organ culture, as the bones cultured in negative scrum with ApoE reached length similar to those cultured in positive serum (Fig. 5C). These results indicated that ApoE has potential to stimulate not only chondrocyte proliferation, but also bone growth, at least in vitro. Furthermore, ELISA for ApoE using bone extracts revealed that the amount of ApoE per femur was significantly reduced in OI model mice compared to wild type mice (Fig. 5D). Together with a report by Massaro et al. demonstrating that ApoE knockout mice have smaller and shorter body size compared to wild type mice, these data suggest that, in OI mice, lower ApoE level around the growth plate hinders chondrocyte proliferation, leading to shorter bones. Massaro et al., Lung Cellular and Molecular Physiology, 294, L991-L997 (2008). MSC infusion might improve bone growth by elevating ApoE levels and stimulating chondrocyte proliferation in the growth plate. Thus, these results strongly suggest that ApoE is the chondrocyte factor released into the serum following MSC infusion.

Example 6: Growth plate analysis of G610C OI mice.

[0071] G610C OI mice carry a Colla2 mutation, which is the same mutation found in Old Order Amish kindred patients. Daley et al., J Bone Miner Res, 25, 247-261 (2010). This mouse model has been widely used as a faithful model of OI by many investigators in the field. Despite many studies about bone quality, phenotypes of this strain related to bone growth have not been characterized well. Thus, bone length was first examined in this strain at 3 -weeks, 6-weeks, and 12- weeks of age in comparison with age-matched wild type mice. Femora of these mice were significantly shorter than those of wild type mice at any age examine (Fig. 6A). The growth plate was then examined histologically. Consistent with the findings in OI patients, both growth plate height and hypertrophic zone (HZ) height were significantly elongated in this OI model compared to age-matched wild type mice (Figs. 6B- 6C), validating that this is an appropriate model to study the growth impairment of OI.

Example 7: In vitro hypertrophic differentiation.

[0072] The prolonged HZ height indicates that the terminal differential of hypertrophic chondrocytes is suppressed. To verify this, the primary chondrocyte pellet culture was utilized as an in vitro hypertrophic differentiation model introduced by Dr. Warman's group. Bowen et al., PLoS Genetics 10, el 004364 (2014). In this culture system, the proliferative chondrocytes in the pellet transit to hypertrophic chondrocytes in 3 weeks, and the expression of osteoblast related genes, which are known to be expressed by terminally differentiated hypertrophic chondrocytes, is increased. While there were no significant differences in expression levels between wild type and OI pellets at 1 week, the expression levels were significantly lower in OI pellets after 3 -weeks of culture, suggesting that terminal hypertrophic differentiation is suppressed in OI chondrocytes (Fig 7 black bars). Since ApoE stimulated chondrocyte proliferation, using this system, the inventors examine whether ApoE could also stimulate differentiation. When ApoE was added into the medium (gray bars), the expression levels of all tested genes except Coll and BGLAP were increased in both wild type and OI pellets, although not all of the changes reached statistical significance. Moreover, ApoE stimulated the expression of Runx2, OPN, ALP and BSP in OI pellets up to the levels observed in wild type pellets. These results suggest that ApoE stimulated differentiation in addition to proliferation of chondrocytes.

Example 8: Treatment Studies

[0073] A variety of treatment studies were carried out to evaluate the effect of ApoE treatment in mice. The results of a loss-of-function study are shown in Figure 9. To verify that ApoE is the chondrocyte factor, the inventors performed "loss-of-function" study using ApoE knockout mice (ApoE KO mice). In response to MSC infusion, WT mice and OI mice released chondrocyte factor into the serum and the sera from these mice stimulated chondrocyte proliferation in the chondrocyte proliferation assay. However, the serum from MSC-infused ApoE KO mice failed to stimulate chondrocyte proliferation, indicating that ApoE is a critical mediator in MSC-induced bones growth.

[0074] The treatment study in which OI was treated by administration of MSC is shown in Figure 10. G610C OI mice were treated with MSC. Specifically, G610C OI mice were infused with either 1 x 10⁶ MSC or PBS once a week for 4 weeks from 3-week of age, then x- ray photos were obtained at 8-week of age. At 3-week old prior to the treatment, there were no significant differences in body weight, femur and tibia length between PBS and MSC groups. However, as observed in patients with OI, MSC infusion significantly stimulated bone growth both in the femur and tibia. [0075] MSC therapy for OI mice lacking ApoE gene is shown in Figure 11. If ApoE is the chondrocyte factor, OI mice lacking ApoE gene (G610C OI/ApoE KO double mutant mice) should not respond to MSC infusion. To confirm this, G610C OI/ApoE KO mice were treated with MSC as performed to G610C OI mice in Figure 10. Contrary to G610C OI mice, G610C OI mice lacking ApoE gene did not respond to MSC therapy and no significant difference was observed between PBS and MSC groups. These results also support that ApoE is necessary to provide MSC therapeutic effects on bone growth.

[0076] Recombinant ApoE therapy for OI mice is shown in Figure 12. The inventors' data strongly indicate that chondrocyte factor is ApoE. They therefore treated G610C OI mice with recombinant ApoE (recombinant human ApoE3). To maintain the serum levels of ApoE in the treating animals, osmotic pumps which release the contents at a constant rate for 7 days were used. Specifically, the inventors subcutaneously implanted an osmotic pump containing either PBS or 170 μg of recombinant ApoE at 3 -week of age, and then replace the pump once a week 3 times (28 days treatment). Bone length was evaluated at 8-week old by x-ray. X-ray analyses revealed that ApoE treatment stimulated bone growth both in the femur and tibia.

[0077] Collectively, these results confirmed that ApoE is a critical mediator of MSC-induced bone growth in OI and recombinant ApoE can be used as a pharmaceutical drug to treat growth deficiency in OI.

[0078] The complete disclosure of all patents, patent applications, and publications, and electronically available material cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art wilt be included within the invention defined by the claims.

Claims

CLAIMS What is claimed is:

1. A method of stimulating bone growth in a subject, comprising administering a therapeutically effective amount of Apolipoprotein E (Apo E), an analog or homolog thereof, or an Apo E mimetic peptide, to the subject.

2. The method of claim 1, wherein the subject has been diagnosed as having a bone growth disease or disorder.

3. The method of claim 2, wherein the subject has been diagnosed as having bone growth disease or disorder selected from the group consisting of osteogenesis imperfecta, disorders caused by increased osteoclastogenesis or bone loss associated with inflammatory conditions, infection, genetic and age-related bone disorders such as osteoporosis, osteopenia, Paget's disease, metastatic bone cancer, myeloma bone disease, bone fracture healing, and bone graft repair.

4. The method of claim 2, wherein the subject has been diagnosed as having osteogenesis imperfecta.

5. The method of claim 1, wherein the subject has been diagnosed as having an increased risk of developing a bone growth disease or disorder.

6. The method of claim 1, wherein Apo E or an analog or homolog thereof is administered.

7. The method of claim 1, wherein an Apo E mimetic peptide is administered.

8. The method of claim 1, wherein the Apo E, an analog or homolog thereof, or an Apo E mimetic peptide, is administered together with a pharmaceutically acceptable carrier.

9. The method of claim 1, wherein the Apo E, an analog or homolog thereof, or an Apo E mimetic peptide, is administered repeatedly or continuously over a significant period of time.

10. The method of claim 1, wherein the ApoE3 allele isoform of Apo E is administered.

11. The method of claim 1, further comprising administration of one or more additional bone growth stimulating agents to the subject.