Patent ID: 12188049

DETAILED DESCRIPTION OF THE INVENTION

Where the definition of terms departs from the commonly used meaning of the term, applicant intends to utilize the definitions provided below, unless specifically indicated

As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the content clearly dictates otherwise.

As used herein, the use of “or” means “and/or” unless stated otherwise. In the context of a multiple dependent claim, the use of “or” refers back to more than one preceding independent or dependent claim in the alternative only.

As used herein, the term “one or more” is readily understood by one of skill in the art, particularly when read in context of its usage.

As interchangeably used herein, the terms “individual,” “subject,” “host,” and “patient,” refer to a mammal, including, but not limited to, murines (rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc.

As used herein, the term “soluble P-selectin” refers to naturally occurring soluble form of P-selectin and recombinant form thereof, or polymorphic or allelic variant or other isoforms thereof. The term also comprises modified or unmodified soluble P-selectin, such as glycosylated or non-glycosylated forms.

As used herein, the terms “mobilize” and “mobilization” refer to processes by which a population of hematopoietic stem or progenitor cells is released from a stem cell niche.

As used herein, the term “niche” refers to the in vivo or in vitro cellular and molecular microenvironments that regulate stem cell function together with stem cell autonomous mechanisms. This includes control of the balance between quiescence, self-renewal, and differentiation, as well as the engagement of specific programs in response to stress.

As used herein, the term “hematopoietic stem cell” or “HSC” refers to a stem cell that is capable of differentiating into both myeloid lineages (i.e. monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets and some dendritic cells) and lymphoid lineages (i.e. T-cells, B-cells, NK-cells, and some dendritic cells).

As used herein, the term “subject” refers to any animal, including mammals, birds, reptiles and amphibians and in preferred embodiments to mammals, including humans, companion animals, food production animals and wild animals.

As used herein, the term “donor” refers to a subject from which one or more cells are isolated prior to administration of the cells, or progeny thereof, into a recipient.

As used herein, the term “effective amount” refers to a quantity of one or more agents, such as a quantity of a soluble P-selectin and/or a second agent described herein that mobilizes a population of hematopoietic stem or progenitor cells upon administration to a subject.

As used herein stem-cell niche (adult or fetal) refers to a microenvironment, within the specific anatomic location where stem cells are found, which interacts with stem cells to regulate cell fate.

Stem cells can produce new cells to repair damage to tissues and therefore have great potential for regenerative medicine. However, they exist in small quantities in tissues and especially in peripheral blood, making it difficult to collect them or use them clinically. Mobilization of stem cells is a way to collect stem cells from bone marrow into the blood. The present disclosure sparingly found that a soluble P-selectin (sP-sel) may interfere in the interaction between stem cells and niches and thus mobilize stem cells from bone marrow. Accordingly, the mobilization of stem cells with sP-sel can treat a subject in need of one or more of preservation, repair, or regeneration of a tissue, or revascularization in the subject.

P-selectin is a member of the selectin family localized in the membranes of α-granules of platelets and the Weibel-Palade bodies (WP bodies) of endothelial cells. P-selectin is expressed as two different forms; one is the “cell-surface” form and the other is the “soluble” form. The former one is expressed on the activated platelet or endothelial cell that is involved in the inflammation of leukocytes and the homing of HSCs. The latter one (i.e., sP-sel) is almost exclusively expressed in the plasma only during the animal/human, which is under stress (e.g. hypoxia) (Chang, H. H. & Sun, D. S. Methods of reducing hypoxic stress in a mammal by administering soluble P-selectin. U.S. Pat. No. 8,377,887 B1 (2012)). A soluble P-selectin molecule, which exists as a monomer in the blood, is 3 kDa smaller than a P-selectin molecule, which exists as an oligomer on a membrane. The soluble P-selectin of healthy individuals originates from the alternatively spliced form found in endothelia cells and platelets. The present invention surprisingly found that treatment of soluble P-selectin may interfere in the interaction between stem cells and niches and thus mobilizes stem cells. The hematopoietic stem and progenitor cells thus mobilized may then be withdrawn from the donor and administered to a patient, where the cells may home to a hematopoietic stem cell niche and re-constitute a population of cells that are damaged or deficient in the patient.

Any forms of sP-sel suitable for mobilization of stem cells can be used in the present disclosure. Examples of the sP-sel of the present disclosure include, but are not limited to, naturally occurring sP-sel and recombinant sP-sel. The sP-sel can be easily obtained by general techniques, such as being isolated from a natural source, purchased from a commercial source, or synthesized with molecular biological techniques. As used herein, exemplary p-selectin may also include in the alternative, platelet alpha-granule membrane protein, CD62, granulocyte membrane protein; GRMP; and/or GMP140.

The sP-sel can be used in combination with a second agent for mobilization of stem cells. Examples of the second agent include, but are not limited to, G-CSF, GM-CSF, IL-3, GM-CSF/IL-3 fusion proteins, FLK-2/FLT-3 ligand, stem cell factor, IL-6, IL-11, TPO, VEGF, AMD3100 and combinations thereof. Preferably, the second agent is G-CSF. The sP-sel and the second agent can be used simultaneously or sequentially.

The sP-sel can be used in combination with stem cells. The sP-sel and the stem cells are administered concurrently, separately or intermittently.

Hematopoietic stem cell transplant therapy can be administered to a subject in need of treatment so as to populate or repopulate one or more blood cell types, such as a blood cell lineage that is deficient or defective in a patient suffering from a stem cell disorder. Hematopoietic stem and progenitor cells exhibit multi-potency, and can thus differentiate into multiple different blood lineages. Hematopoietic stem cells give rise to different types of blood cells, in lines called myeloid and lymphoid. Myeloid and lymphoid lineages both are involved in dendritic cell formation. Myeloid cells include monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, and megakaryocytes to platelets. Lymphoid cells include T cells, B cells, natural killer cells, and innate lymphoid cells.

Hematopoietic stem or progenitor cells mobilized to the peripheral blood of a subject can be withdrawn (e.g., harvested or collected) from the subject by any suitable technique. For example, the hematopoietic stem or progenitor cells may be withdrawn by a blood draw. In some embodiments, hematopoietic stem or progenitor cells mobilized to a subject's peripheral blood as contemplated herein may be harvested (i.e., collected) using apheresis. In some embodiments, apheresis may be used to enrich a donor's blood with mobilized hematopoietic stem or progenitor cells.

The sP-sel disclosed herein can be administered according to various routes of administration, typically by injection, such as local or systemic injection(s). However, other administration routes can be used as well, such as intramuscular, intravenous, intradermic, subcutaneous, etc. For administration, the sP-sel is ordinarily combined with one or more adjuvants appropriate for the indicated route of administration. Furthermore, repeated injections can be performed, if needed. The sP-sel is administered in the range of approximately 10−5μg to 1.5 mg per kg body weight.

The sP-sel-mobilized circulating stem cells (PselMSCs) can ameliorate tissue or organ damage, increase repair, improve glucose tolerance and/or reduce inflammation. The PselMSCs also can repopulate the bone marrow or hematopoietic stem cell population and rescue tissue injuries, proliferative disorders, inflammatory diseases, immunodeficiency diseases, genetic disorders, degenerative disorders, autoimmune disorders and/or metabolic diseases. Examples of proliferative disorders include, but are not limited to, hematologic cancer and myeloproliferative disease. Example of immunodeficiency diseases include, but are not limited to, congenital immunodeficiency diseases and acquired immunodeficiency diseases. Example of autoimmune disorders include, but are not limited to, juvenile arthritis, ulcerative colitis, Type 1 diabetes mellitus (Type 1 diabetes), multiple sclerosis (MS), inflammatory bowel disease (IBD), psoriasis, psoriatic arthritis, rheumatoid arthritis (RA), human systemic lupus (SLE), autoimmune lymphoproliferative syndrome (ALPS), and lymphocytic colitis. Example of metabolic diseases include, but are not limited to, glycogen storage diseases, mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease, sphingolipidoses, and metachromatic leukodystrophy.

Withdrawn hematopoietic stem or progenitor cells may be re-infused into the patient, such that the cells may subsequently home hematopoietic tissue and establish productive hematopoiesis, thereby populating or repopulating a line of cells that is defective or deficient in the patient.

Although the invention has been described with reference to preferred embodiments and examples thereof, the scope of the present invention is not limited only to those described embodiments. As will be apparent to persons skilled in the art, modifications and adaptations to the above-described invention can be made without departing from the spirit and scope of the invention, which is defined and circumscribed by the appended claims. The following examples are provided for the intent of illustrating embodiments and advantages of the invention and are not intended to limit its scope.

EXAMPLE

Example 1 Soluble P-Selectin Mediated Mobilization of CD34+ Cells

According to a previous report (Tajima, F., Sato, T, Laver, J. H. & Ogawa, M CD34 expression by murine hematopoietic stem cells mobilized by granulocyte colony-stimulating factor.Blood96, 1989-1993 (2000)), G-CSF-mediated mobilization of mouse CD34+cells needs 5 doses of injections. Here we showed that on day 3 (after 2 doses of G-CSF injections), there are still no elicitation of circulating CD34+cells in mice.

The recombinant mouse P-selectin (rmP-sel) and granulocyte-colony stimulating factor (G-CSF; Filgrastim®) were injected intravenously per treatment (0.1 mg/kg body weigh) to mice. Compared to no induction of circulating CD34+ stem cells previously, the rmP-sel treatments surprisingly and unexpectedly elicited significant amount of CD34+ cells with only 2 injections (seeFIG.1Aexperimental outline, 1B). These results show that P-selectin is much potent than G-CSF on the mobilization of circulating CD34+ cells.

Example 2 Soluble P-Selectin Ameliorated Thioacetamide (TAA)-Induced Thrombocytopenia and Liver Injury

C57BL/6J mice were injected with TAA to induce acute liver injury and then rescued with or without rm-Psel (FIG.2A, time table). We found that pretreatment with twice of rmP-sel could rescue reduced-platelet counts in peripheral blood to normal level (FIG.2B). Aspartate aminotransferase (AST) enzyme activity is a standard marker to measure liver injury. Data also revealed that rmP-sel ameliorated the liver damages (FIG.2C). Our data suggested that rmP-sel treatments exert tissue protective effect to reduce TAA-induced injury (n=4; results with statistical significance, P<0.05 rmP-sel vs. saline).

It has previously shown that stem cell treatments might have beneficial effects on the amelioration on the liver damages. To characterize whether rmP-Sel-elicited CD34+cells have tissue protective effect, we performed the adoptive transfer experiment. Our data showed that adoptive transfer of P-selectin-mobilized CD34+stem cells, but not peripheral blood mononuclear cells (PBMCs) is able to rescue TAA-induced hepatitis in mice (FIG.3).

In human, G-CSF treatments mobilize CD34+stem cells, which are able to repopulate the γ-irradiated bone marrow. The LSK hematopoietic stem cell in mice is the cell lineage equivalent to the CD34+stem cell in human. To investigate the potential role of P-selectin-mobilized LSK cells in the repopulation of bone marrow, C57Bl/6 recipient mice that had received lethal irradiation with γ-ray were transplanted with 1×105LSK cells mobilized with soluble P-selectin (FIG.4) and G-CSF (FIG.5). The engraftments are successfully transplanted as revealed by the rescue of 100% lethal γ-irradiation in mice (FIG.5; 100% lethal in non-transplanted groups vs. approximately 83% survival of both soluble P-selectin mobilized LSK and G-CSF mobilized LSK groups).

Protective role of PselMSCs and PselSCMVs, soluble P-selectin and soluble P-selectin-conjugated liposome were demonstrated using the TAA hepatitis mouse model. Treatments of PselMSCs and PselSCMVs, soluble P-selectin and soluble P-selectin-conjugated liposome markedly rescued TAA-induced elevation of high circulating alanine aminotransferase (ALT) levels in mice, suggesting that these reagents have ameliorative effects on TAA-induced liver damages (FIG.6).

It is previously shown that adipose-derived mesenchymal stem cells may improve glucose homeostasis in high-fat diet-induced obese mice. However, whether PselMSCs, PselSCMVs, soluble P-selectin and soluble P-selectin-conjugated liposome remains elusive. Results of glucose tolerance (OGTT) analysis conclusively demonstrated that PselMSCs, PselSCMVs, soluble P-selectin and soluble P-selectin-conjugated liposomes all ameliorated high fat diet (HFD)-induced glucose resistant, whence reduce the blood glucose levels of HFD-mice (FIG.7).

Circulating TNF-α levels, which reveal the degree of inflammation, were analyzed in TAA-induced hepatitis mice. Data revealed that PselMSCs, PselSCMVs, soluble P-selectin and soluble P-selectin-conjugated liposomes all contain anti-inflammatory property, as treatments of these agents all markedly reduced TAA-induced induction of plasma TNF-α levels in mice (FIG.8).