Compositions using iron excipients and uses thereof, including cancer treatment

Abstract

A bone graft composition is provided that includes a calcium phosphate putty. A method of applying the bone graft composition to repair a bone defect in a patient is also provided.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 053,277, filed July 17, 2020, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Bone grafting is a surgical procedure performed to repair fractures that pose significant health risks to the patient or that do not heal properly. Certain small or acute fractures can be healed, but larger fractures such as compound fractures pose a higher risk. Bone generally has the ability to regenerate completely, but only if there is a very small fracture space or some sort of scaffolding. Successful bone grafts can result in osteoconduction (the ability of the bone graft material to passively allow bone growth), osteoinduction (the bone graft induces undifferentiated cells to become active osteoblasts that form new bone tissue), and / or osteogenesis (living bone cells in the graft material contribute to bone remodeling).

[0003] Bone grafts are made from autologous bone, i.e., the bone graft material is harvested from the patient's own body, often from the iliac crest. However, the use of autologous bone subjects the patient to increased pain and discomfort, and an increased risk of infection, as the patient must undergo additional surgery to retrieve the autologous bone for use in the graft procedure.

[0004] The bone graft may be an allograft, i.e., the bone graft material is derived from cadaveric bone, usually obtained from a bone bank. Allograft bone exposes the patient to risk of disease and rejection of the graft.

[0005] Bone graft materials may be synthetic, and synthetic bone graft materials are often made of hydroxyapatite or other naturally occurring biocompatible materials with mechanical properties similar to those of bone. Most bone grafts are expected to be resorbed and replaced over a period of months as the natural bone heals. However, synthetic bone graft materials may be less osteoconductive or osteoinductive than autograft or allograft materials. Synthetic materials may also be susceptible to donor microbial infection. Thus, there is a great medical need for synthetic bone graft materials with improved properties for healing bone injuries. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] In one aspect, a bone graft composition is provided that includes a calcium phosphate putty and at least one of a hardening agent, a hardening rate control agent, an acidifying agent, an iron excipient, collagen, and a diluent solution.

[0007] In some embodiments, the calcium phosphate putty comprises biphasic calcium phosphate particles. In some embodiments, the biphasic calcium phosphate particles comprise hydroxyapatite and tricalcium phosphate. In some embodiments, the biphasic calcium phosphate particles comprise about 20-60% hydroxyapatite and about 40-80% tricalcium phosphate. In some embodiments, the biphasic calcium phosphate particles have interconnected macropores and micropores. In some embodiments, the biphasic calcium phosphate particles are in the form of spherical particles, fibers, or irregular granules. In some embodiments, the calcium phosphate putty comprises biphasic calcium phosphate particles, the biphasic calcium phosphate particles comprise hydroxyapatite and tricalcium phosphate, and the bone graft composition further comprises collagen and naloxone.

[0008] In some embodiments, the bone graft composition further comprises a bioabsorbable polymer.

[0009] In some embodiments, the bone graft composition has a density of 1-6 g / mL after mixing. In some embodiments, the force required to extrude the bone graft composition through a cannula, tube, or syringe is less than 80 N / m.

[0010] In some embodiments, the bone graft composition does not have an active agent. In some embodiments, the bone graft composition further comprises an active agent. In some embodiments, the bone graft composition is constructed and arranged to deliver an active agent or other agent to a desired site in a patient.

[0011] In some embodiments, the bone graft composition comprises a network of reservoirs and microchannels for storing and delivering an active agent. In some embodiments, the bone graft composition comprises a plurality of reservoirs, microtubes and / or nanotubes for storing and delivering an active agent. In some embodiments, the active agent is delivered at a controlled rate.

[0012] In some embodiments, the active agent is dissolved in a pharma- ceutically suitable carrier. In some embodiments, the active agent dissolved in a pharma- ceutically suitable carrier is sprayed or coated onto the synthetic bone graft.

[0013] In some embodiments, the active agent is an opioid growth factor receptor (OGFR) antagonist. In some embodiments, the OGFR antagonist is selected from the group consisting of naloxone, naltrexone, and salts thereof. In some embodiments, the OGFR antagonist is administered with a diluent.

[0014] In some embodiments, the bone graft composition comprises a hardening agent, and the hardening agent is sodium carbonate (NaCO 3 ) or iron sulfate (FeSO 4In some embodiments, the bone graft composition comprises a hardening agent, which comprises one or more compounds from the group consisting of calcium sulfate hemihydrate, calcium sulfate dihydrate, calcium sulfate anhydrous, monocalcium phosphate, dicalcium phosphate, tricalcium phosphate, tetracalcium phosphate, octacalcium phosphate, hydroxyapatite, calcium carbonate, magnesium carbonate, strontium carbonate, and sodium hydrogen phosphate.

[0015] In some embodiments, the weight ratio of the hardening agent to the calcium phosphate salt compound is between 1:2 (hardening agent:calcium salt) and 3:4 (hardening agent:calcium salt).

[0016] In some embodiments, the bone graft composition comprises an acidifying agent, the acidifying agent comprising one or more from the group of ascorbic acid, magnesium citrate, potassium citrate, sodium citrate, citric acid monohydrate, and acetic acid.

[0017] In some embodiments, the acidifying agent increases the bioavailability of the active agent in the bone graft composition. In some embodiments, the acidifying agent promotes bone formation. In some embodiments, the acidifying agent increases the adhesiveness of the bone graft material.

[0018] In some embodiments, the bone graft composition comprises a color forming agent. In some embodiments, the color forming agent comprises an activatable color forming agent. In some embodiments, the color forming agent colors the bone graft composition red, blue, orange, green, neon green, purple, brown, black, gray, or battleship gray.

[0019] In some embodiments, the bone graft composition comprises an antimicrobial agent. In some embodiments, the antimicrobial agent comprises one or more from the group of antibiotic agents or antifungal agents.

[0020] In some embodiments, antibiotics include vancomycin, gentamicin, tobramycin, kanamycin, neomycin, ampicillin, methicillin, nafcillin, oxacillin, penicillin, ticarcillin, ciprofloxacin, vancomycin, cefazolin, cefepime, ceftriaxone, clindamycin, aztreonam, imipenem, quinupristin / dalfopristin, chloramphenicol, doxycycline, metronidazole, nitrofurantoin, polymyoxin B, tetracycline, viomycin, chloromycetin, streptomycin, azactam, and pharmaceutically acceptable salts thereof, and combinations thereof.

[0021] In some embodiments, the antifungal agent comprises one or more of the group of polyene antifungals, imidazoles, triazoles, allylamines, and echinocandins. In some embodiments, the bone graft composition exhibits antibacterial and / or antifungal efficacy. In some embodiments, the bone graft composition is not a dental resin.

[0022] In another aspect, there is provided a method of repairing a bone defect in a patient in need thereof, the method comprising applying a bone graft composition disclosed herein to the bone defect.

[0023] In some embodiments, the bone defect is caused by a human disease or condition, and wherein the bone graft composition includes an active agent for treating the human disease or condition. In some embodiments, the bone defect is caused by a human disease or condition, and wherein the bone graft composition does not contain an active agent for treating the human disease or condition. In some embodiments, the human disease or condition is one of several disorders of the vertebrae of the spine, which require a surgical procedure to fuse the vertebrae together. In some embodiments, the human disease or condition is one of several disorders of the bones of the limbs, which require a surgical procedure to fuse the bones together or to repair the defect. In some embodiments, the human disease or condition is a void or defect in bone that is not essential to the stability of the bone structure, and the void or defect in bone is caused by surgery or traumatic injury, and the bone graft composition fills the void or defect in bone. In some embodiments, the bone defect comprises a void or gap in the subject's skeletal system, the void or gap is not essential to the stability of the skeletal structure, and the bone graft composition is to fill the void or gap.

[0024] In some embodiments, the surgical procedure includes a lumbar interbody fusion (LIF) procedure. In some embodiments, the LIF procedure includes anterior LIF, lateral LIF, transforaminal LIF, and posterior LIF. In some embodiments, the LIF procedure alleviates associated symptoms, such as the subject's degenerative disc disease as determined via radiography, resulting from the formation of bone defects via the surgical procedure. In some embodiments, the LIF procedure alleviates associated neuromuscular or physical deficits in the subject as determined by the subject's Oswestry Disability Index (ODI), the subject's neurological assessment, or the subject's radiography.

[0025] In some embodiments, the human disease or condition is cancer. In some embodiments, the bone defect is caused by cancer. Specific types of cancer include, but are not limited to, skin cancer (e.g., melanoma), connective tissue cancer (e.g., sarcoma, osteosarcoma, Ewing's osteosarcoma, giant cell carcinoma), breast cancer, head and neck cancer, lung cancer (e.g., non-small cell lung cancer), gastric cancer, pancreatic cancer, ovarian cancer, cervical cancer, uterine cancer, anogenital cancer (e.g., testicular cancer), kidney cancer, bladder cancer, colon cancer, prostate cancer, central nervous system (CNS) cancer (e.g., neuroblastoma, etc.), retinal cancer, blood cancer (e.g., multiple myeloma, etc.), and lymphatic system cancer (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma).

[0026] In some embodiments, the bone defect is caused by a malignant tumor or surgical removal of a malignant tumor via resection. In some embodiments, the malignant tumor comprises a breast cancer metastasis. In some embodiments, the methods disclosed herein use the disclosed bone graft compositions for reconstruction of the metaphysis-diaphysis of the humerus of a subject following resection of a malignant tumor, such as a breast cancer metastasis, by use of an intercalary allograft and plate fixation.

[0027] In some embodiments, the bone graft composition is osteoconductive. In some embodiments, the bone graft composition is osteoinductive. In some embodiments, the bone graft composition is osteopromotive. [Brief description of the drawings]

[0028] [Figure 1]MC3T3-E1 subclone 4 mouse calvaria-derived bone-forming cells were cultured as spheroids (micro-clusters) to become osteoblasts. Cultures were either untreated (negative control), chemically treated to induce differentiation into osteoblasts (positive control), or cultured with bone graft material containing ferrous sulfate vehicle (ferrous sulfate bone graft). Cultures were grown for 7 days before adding bone graft material or chemical induction reagent. After an additional 12 days, cultures were stained for mineral deposition with Alizarin Red stain (dark stain) (Figure 1A). The ferrous sulfate cultures had significantly more staining than either control group. Cultures were then assessed for the amount of mineral contained in the spheroids to determine if mineral had increased (Figure 1B). The ferrous sulfate bone graft cultures had significantly more mineral in the spheroids versus the negative control cultures (* = p<0.0001). Minerals in the positive control culture medium were not significantly different from those in the negative control culture medium (ns = not significant). Summary of the Invention

[0029] Described herein are compositions, materials, methods, and kits for bone grafting and repairing and / or filling voids or gaps in bone. The present disclosure provides bone graft compositions with improved material properties and improved bioavailability of drugs contained in the materials. The present disclosure also provides bone graft compositions with coloring agents that allow for easy and convenient detection of the implanted material. Additionally, included herein are bone graft compositions with improved antimicrobial functionality. Finally, the present disclosure provides bone graft compositions containing active agents with therapeutic properties for treating diseases and conditions in which it is desirable or necessary to promote bone growth, either by stimulating bone formation or preventing bone destruction.

[0030] I. Definition To facilitate understanding of certain terms used herein, the following definitions are provided.

[0031] Technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art unless otherwise defined. Any suitable materials and / or methodologies known to those of ordinary skill in the art may be utilized in carrying out the methods described herein.

[0032] As used in the description of this invention and the appended claims, the singular forms "a," "an," and "the" are used interchangeably and are intended to include the plural forms as well and to be consistent with their respective meanings, unless the context clearly indicates otherwise. Also, as used herein, "and / or" refers to and includes all possible combinations of one or more of the listed items, as well as the lack of combination when interpreted in the alternative ("or").

[0033] All numerical designations, e.g., pH, temperature, time, concentration, amount, and molecular weight, including ranges, are approximations which vary by ±10%, 1%, or 0.1%, as appropriate. It is understood, although not always expressly stated, that all numerical designations may be preceded by the term "about." It is also understood, although not always expressly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0034] The terms "comprising" or "including" are intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding other elements that are essential to the combination. For example, a composition consisting essentially of the elements defined herein would not exclude other elements that do not materially affect the basic and novel property(ies) of the claimed invention. "Consisting of" is intended to mean excluding more than trace amounts of other ingredients and substantial method steps as described. Embodiments defined by each of these transitions are within the scope of the invention.

[0035] As used herein, the term "bone graft composition" refers to any malleable composition suitable for repairing bone defects. The bone graft composition may be a "calcium phosphate putty." In some embodiments, the calcium phosphate putty is a bone void filler.

[0036] As used herein, the term "active agent" means any entity, including a chemical or biological entity, approved by a regulatory agency, e.g., the USFDA, for the treatment of a particular indication.

[0037] The term "active agent free" in the context of a composition or formulation means a composition or formulation that does not contain an active agent, as defined herein.

[0038] As used herein, the term "biocompatibility" refers to the ability (e.g., of a composition or material) to perform in a particular application with an appropriate host response, or at least to perform without toxic or other detrimental effects, locally or systemically, on the host's biological system.

[0039] As used herein, the term "osteoconductive" refers to the ability (e.g., of a composition or material) to passively (e.g., onto and / or into) bone growth. As such, osteoconduction can be characterized as a passive process. Osteoconductive materials or compositions will only contribute to new bone growth in areas where significant bone already exists.

[0040] A material (e.g., a graft or implant) can be osteoconductive, for example, because it is configured to passively allow bone growth onto a surface of the material. In another example, a material can be osteoconductive because it is configured to passively allow bone growth into openings (e.g., pores) in the material.

[0041] The term "osteoinductive" as used herein refers to the ability (e.g., of a composition or material) to actively stimulate a biological response that induces bone formation. Thus, osteoinduction can be characterized as an active process. Osteoinductive materials or compositions can induce new bone growth and contribute to new bone growth in areas that lack significant bone.

[0042] Osteoinductivity may include the formation and / or stimulation of bone precursor cells, for example, bone precursor cells in body tissues surrounding or adjacent to the graft or implant.

[0043] As used herein, the term "osteopromoting" refers to the ability (e.g., of a composition or material) to promote new bone formation by enhancing the osteoinductivity of an osteoinductive material. An osteopromoting composition or material enhances osteoinductivity, but is not inherently osteoinductive.

[0044] As used herein, the term "bioactive" refers to the ability (eg, of a composition or material) to form a hydroxyapatite (HA) surface layer when submerged in simulated body fluid (SBF).

[0045] As used herein, the term "osteostimulatory" refers to the ability (e.g., of a composition, material, or extract thereof) to enhance or positively stimulate osteoblast proliferation and mesenchymal stem cell differentiation.

[0046] As used herein, the terms "antimicrobial" and "antibacterial" refer to the U.S.P. <51> "Inhibition of microbial growth" refers to the ability (e.g., of a composition, material, or extract thereof) to inhibit the growth of microorganisms according to the methods described above.

[0047] As used herein, the term "biodegradable" refers to the ability of a material to be degraded, broken down, and / or digested over time by the action of a biological environment (including the action of an organism, e.g., a patient's body) and / or in response to changes in physiological pH or temperature. In the context of the human body environment, biodegradable means that the material will degrade, break down, and / or be digested under normal physiological conditions.

[0048] As used herein, the terms "resorbable" and "bioabsorbable" refer to the ability of a material to be broken down over a period of time and assimilated into the body environment. Resorbable and bioabsorbable in the context of the human body environment means that the material will be broken down over a period of time and assimilated into the body under normal physiological conditions.

[0049] As used herein, the term "moldable" means the property of being flexible and capable of being manually compressed, molded, and manipulated while maintaining integrity, compositional homogeneity, physical properties, and performance characteristics.

[0050] As used herein, a reference to the weight of a component of a bone graft composition or material described herein, such as the phrase "by weight," means the weight of the corresponding component prior to being added to or mixed with another different component of the bone graft composition. For example, the weight can refer to the initial weight of the component measured prior to further processing of the component into a bone graft composition.

[0051] As used herein, the expression "non-weight-bearing applications" refers to applications for the repair of a void or gap in a bone or another bony structure, where the void or gap being repaired is not essential to the stability of the bone or bony structure.

[0052] As used herein, the term "antagonist" is used interchangeably with "inhibitor" and refers to a substance that blocks or inhibits the activity, function, effect, or expression of a target. In some embodiments, the target is a compound, a protein, a gene, a cell, or a drug. The term "expression" as used herein means the amount of a target produced by a living cell. In some embodiments, an inhibitor suppresses the expression of a target gene or protein. In some embodiments, an inhibitor includes a compound that blocks the binding of another molecule to an enzyme or molecular pump. In some embodiments, an inhibitor is a compound that causes downregulation of an enzyme. In some embodiments, an inhibitor can be a competitive inhibitor or a non-competitive inhibitor.

[0053] As used herein, the term "administering" includes formulating for administration as well as actually administering, including physically administering by the subject being treated or another.

[0054] As used herein, "subject," "patient," or "individual" refers to any subject, patient, or individual, and the terms are used interchangeably herein. In this regard, the terms "subject," "patient," and "individual" include mammals, particularly humans, dogs, and cats. When used in connection with "in need thereof," the term "subject," "patient," or "individual" contemplates any subject, patient, or individual having or at risk for a particular condition or disorder.

[0055] As used herein, the phrase "therapeutically effective" or "effective" in the context of "dosage" or "amount" refers to a dosage or amount that provides the particular pharmacological effect for which the compound or compound is administered. It is emphasized that a therapeutically effective amount is not always effective in achieving the intended effect in a given subject, even if such a dosage is considered to be a therapeutically effective amount by those skilled in the art. For convenience only, exemplary dosage amounts are provided herein. Those skilled in the art can adjust such amounts according to the methods disclosed herein to treat a particular subject suffering from a particular condition or disorder. A therapeutically effective amount may vary based on the route of administration and dosage form.

[0056] The term "treat" or "treatment" refers to the treatment of conditions that cause or are associated with bone defects. For example, cancer. Such treatment includes (i) inhibiting cancer, i.e., preventing its development; (ii) alleviating cancer or disorders, i.e., causing regression of cancer; (iii) slowing the progression of cancer; and / or (iv) inhibiting, alleviating, or slowing the progression of one or more symptoms of cancer in a subject, such as a human or animal patient. For example, treating cancer includes, but is not limited to, eliminating cancer or a condition caused by cancer, remission of a tumor, suppressing cancer, or reducing or eliminating at least one symptom of a tumor. Other examples of conditions that cause or are associated with bone defects include, for example, diabetes, osteoporosis, lupus, rheumatoid arthritis, hyperthyroidism, celiac disease, asthma, and multiple sclerosis. Active agents for treating any of the above conditions are well known to those skilled in the art and are within the scope of the present disclosure.

[0057] The term "treat" or "treatment" can also cover the treatment by surgical procedures of conditions that cause or are associated with skeletal biomechanical instability, age-related degenerative conditions, or bone defects due to trauma. The patient can be a human or veterinary patient. For example, spinal fusion due to disc degeneration. Such treatments include, in a human subject, removing the degenerated disc, placing an intervertebral spacer or "cage" between two vertebral segments of the treatment segment, placing bone graft material into the spacer / cage, and then mechanically stabilizing the spine using metal rods and screws. After several months, the bones of the vertebral segments fuse together, resulting in a mechanically stable vertebral segment. Other conditions include fractures, osteotomies, arthroplasties, reconstructive procedures, oral augmentations (i.e., any dental restoration including ridge augmentation, sinus lift, or socket restoration), and defect (e.g., void filler) repair. In some embodiments, the bone defect comprises a void or gap in the bony skeletal system of a subject, where the void or gap is not essential to the stability of the bony skeletal structure. The void or gap in the bony skeletal system of a subject may be treated by gently packing the void or gap with a bone graft composition.

[0058] The condition may also be a disorder of a bone of an extremity, where the bone of the extremity requires a surgical procedure to fuse the bone or repair the defect. In some embodiments, the human disease or condition is a bone void or defect that is not essential to the stability of the bone structure, where the bone void or defect is created by surgery or traumatic injury, and the bone graft composition fills the bone void or defect. In some embodiments, the human disease or condition is a bone void or defect that is not essential to the stability of the bone structure, where the bone void or defect is created by surgery or traumatic injury. In some embodiments, the surgical procedure comprises a lumbar interbody fusion procedure (LIF) procedure. In some embodiments, the LIF procedure comprises anterior LIF, lateral LIF, transforaminal LIF, and posterior LIF. In some embodiments, the LIF procedure alleviates the associated symptoms, for example, intervertebral disc degeneration in the subject, as determined via radiography, due to the formation of a bone defect via a surgical procedure. In some embodiments, LIF treatment ameliorates the subject's associated neuromuscular or physical deficits as determined by the subject's Oswestry Disability Index (ODI), a neurological assessment of the subject, or an x-ray of the subject.

[0059] The term "analog" refers to a compound in which one or more individual atoms or functional groups have been replaced with either different atoms or different functional groups, generally resulting in a compound with similar properties. In some embodiments, an analog refers to a structure that is similar to another, but differs in one or two components.

[0060] The term "derivative" refers to a compound formed from a similar starting compound by attaching another molecule or atom to the starting compound. Additionally, a derivative according to the present invention encompasses one or more compounds formed from a precursor compound by the addition of one or more atoms or molecules, or by the combination of two or more precursor compounds.

[0061] II. Calcium Phosphate Bone Graft Compositions The bone graft compositions or materials according to the embodiments promote bone repair or regeneration at a target repair site. For example, in some embodiments, the bone graft compositions can be osteoconductive, osteoinductive, bioactive, osteostimulatory, antimicrobial, or any combination thereof. The target repair site can be, for example, a void, gap, or other defect in a bone or other bone structure within a patient's body. For example, as described in more detail below, the bone graft compositions promote bone growth at a target repair site in the spine, pelvis, limbs, skull, or other bone or bone structure within a patient's body. The bone graft compositions can be implanted, extruded, molded, or otherwise placed at the target repair site. For example, in some embodiments, the bone graft compositions can be implanted, extruded, molded, or placed at the target repair site in a non-weight-bearing application. In other embodiments, the bone graft compositions can be implanted, extruded, molded, or placed at the target repair site with appropriate orthopedic hardware (i.e., screws, plates, rods, cages / spacers, prostheses, hip implants, knee implants, acetabular implants, etc.) in a load-bearing application.

[0062] In some embodiments, the bone graft composition is a calcium phosphate composition.

[0063] In some embodiments, the calcium phosphate is selected from the following: hydroxyapatite (Ca 5 (OH)(PO 4 ) 3 ), β-tricalcium phosphate (β-Ca 3 (PO 4 ) 2 ), calcium phosphate dibasic (CaHPO 4 ), or calcium phosphate tribasic (Ca 5 (OH)(PO 4 ) 3 ), monocalcium phosphate monohydrate (Ca(H 2 PO 4 ) 2 -H 2 O), dicalcium phosphate dihydrate (CaHPO 4 · 2 H2 O. Octacalcium phosphate (Ca 8 H 2 (PO 4 ) 6 · 5 H 2 O, monocalcium phosphate (Ca(H 2 PO 4 ) 2 ), α-tricalcium phosphate (α-Ca 3 (PO 4 ) 2 ), sintered hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ), oxyapatite (Ca 10 (PO 4 ) 6 O) or tetracalcium phosphate (Ca 4 (PO 4 ) 2 O).

[0064] In some embodiments, the hydroxyapatite has the chemical formula Ca 10-x (HPO 4 ) x (PO 4 ) 6-x (OH) 2-x where "x" can vary between 0 and 2.

[0065] In some embodiments, tricalcium phosphate has the chemical formula Ca 3 (PO 4 ) 2 nH 2 O, where "n" = 3-4.5, H 2 The O content is 15% to 20%.

[0066] In some embodiments, the size of the individual grains (or particles) of the hydroxyapatite material ranges from 1 nm to 5 mm.

[0067] In some preferred embodiments, the particle size for the hydroxyapatite will be selected from the following sizes: 10 μm (microns), 75 μm (microns), 86.4 μm (microns), 125 μm (microns), 147 μm (microns), 212 μm (microns), 368 μm (microns), or 740 μm (microns). In these embodiments, the particle size is understood to represent the maximum size and the range will exist to include potentially smaller particles of material.

[0068] In some embodiments, the individual grains (or particles) of the β-tricalcium phosphate material range in size from 1 nm to 5 mm.

[0069] In some preferred embodiments, the particle size of the β-tricalcium phosphate will be selected from the following sizes: 100 μm (microns), 125 μm (microns), 212 μm (microns), 304 μm (microns), 645 μm (microns), 500 μm (microns), or 1000 μm (microns). In these embodiments, the particle size is understood to represent the maximum size, and the range will include potentially smaller particles of material.

[0070] In some embodiments, the tricalcium phosphate is a mixture of β-tricalcium phosphate and α-tricalcium phosphate.

[0071] In some embodiments, the calcium phosphate salt is anhydrous, monohydrate, dihydrate, or any xH 2 It may be the O hydrate isoform.

[0072] In a preferred embodiment, the calcium phosphate salt included in the preferred formulation of the bone graft material is sintered hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ), β-tricalcium phosphate (β-Ca 3 (PO 4 ) 2 ), Dicalcium phosphate (CaHPO4 ), and tricalcium phosphate (Ca 5 (OH)(PO 4 ) 3 ).

[0073] III. Hardener In some embodiments, the bone graft composition includes a hardening agent. The hardening agent enhances the degree of hardening of the bone graft composition.

[0074] In some embodiments, the hardening agent is sodium carbonate (Na 2 CO 3 ) may also be used.

[0075] In some embodiments, the hardening agent is calcium sulfate anhydrous (CaSO 4 ), calcium sulfate hemihydrate (CaSO 4 -0.5H 2 O), calcium sulfate dihydrate (CaSO 4 - 2 H 2 O), calcium carbonate (CaCO 3 ), Magnesium carbonate (MgCO 3 ), strontium carbonate (SrCO 3 ) and bioglass.

[0076] In some embodiments, the hardener salt may be anhydrous, monohydrate, dihydrate, or any xH 2 It may be the O hydrate isoform.

[0077] In a preferred embodiment, the hardening agent included in the preferred formulation of the bone graft material is sodium carbonate (Na 2 CO 3 ).

[0078] IV. Agents for controlling the hardening rate In some embodiments, the bone graft composition includes a hardening agent. The hardening agent enhances the rate of hardening of the bone graft composition.

[0079] In some embodiments, the hardening agent is calcium oxide (CaO), magnesium oxide (MgO), sodium phosphate dibasic (Na 2 HPO 4 ), Sodium pyrophosphate tetrabasic (Na 4 P 2 O 7 ), Sodium orthophosphate (Na 3 PO 4 ), or monosodium phosphate monobasic (NaH 2 PO 4 ).

[0080] In some embodiments, the sodium phosphate salt may be anhydrous, monohydrate, dihydrate, or any xH 2 It may also include the O hydrate isoform.

[0081] In a preferred embodiment, the setting rate controlling agent is calcium oxide (CaO), magnesium oxide (MgO), and sodium phosphate dibasic (Na 2 HPO 4 ).

[0082] V. Acidifiers It has been discovered by the inventors that the addition of acidifying agents to bone graft compositions results in improved material properties, dissolution, small molecule bioavailability, enhanced bone formation, and improved handling characteristics. Acidifying agents are defined as chemicals, molecules, or ions that can donate a proton (hydrogen ion H+; known as a Bronsted-Lowry acid) or, alternatively, can form a covalent bond with an electron pair (known as a Lewis acid).

[0083] In some embodiments, the bone graft composition comprises an acidifying agent, and the acidifying agent is L-ascorbic acid (C 6 H 8 O 6 ), ascorbic acid 2-phosphate sesquimagnesium salt hydrate (C 12 H 12 O 10 (PO 4 )2 Mg 3 -xH 2 O), sodium citrate dihydrate (Na 3 C 6 H 5 O 7 · 2 H 2 O), magnesium citrate (MgC 6 H 6 O 7 ), potassium citrate monohydrate (K 3 C 6 H 5 O 7 H 2 O), citric acid monohydrate (C 6 H 8 O 7 H 2 O), and acetic acid (C 2 H 4 O 2 ).

[0084] In some embodiments, the acidifier may be anhydrous, monohydrate, dihydrate, or any xH 2 It may be the O hydrate isoform.

[0085] In some embodiments, the acidifying agent is a component of the bone graft material and is a salt or ester species that produces a polyatomic anion that forms in solution and that otherwise fits the rules defining a chemical species as an acid.

[0086] In a preferred embodiment, the acidifying agent is L-ascorbic acid (C 6 H 8 O 6 ), citric acid monohydrate (C 6 H 8 O 7 -H 2 O), and acetic acid (C 2 H 4 O 2 )

[0087] V. Iron Excipients It has been the discovery of the present inventors that the addition of iron excipients in the form of iron salts, iron oxides, iron compounds, iron alloys, and elemental iron to bone graft compositions allows for the following: 1) As a coloring agent, the iron excipient allows for easy detection of where the putty is in relation to the adjacent bone, and allows visualization of proper mixing by evaluating the color uniformity when mixing the liquid and solid precursors of the bone graft composition. 2) Iron excipients modulate the physical properties of bone graft materials by acting as agents that control the rate of hardening and as agents that contribute to hardening. 3) In X-ray imaging (radiography, computed tomography), iron excipients act as contrast agents. 4) Iron excipients contribute to bone formation.

[0088] In some embodiments, the color developing agent comprises a chromogen that is also an iron excipient. As used herein, the term "chromogen" is any chemical species that is an iron excipient that also changes color when dissolved in a solvent.

[0089] In some embodiments, the color former comprises an activatable color former.

[0090] In some embodiments, the coloring agent colors the bone graft composition red, orange, brown, black, or gray. In some preferred embodiments, the coloring agent colors the bone graft composition orange, red, brown, or gray.

[0091] In some embodiments, the iron excipient is ferrous sulfate hydrate (Fe 2 (SO 4 ) 3 ), iron(III) chloride (FeCl 3 ), iron(III) citrate (FeC 6 H 5 O 7 ), iron(III) oxide (Fe 2 O 3 ), iron(III) oxide hydroxide (Fe(OH)O), iron(III) phosphate (FePO 4), ammonium iron(III) citrate (C 6 H 8 O 7 -xFe 3 + -yNH 3 ), elemental iron particles (Fe), iron(III) fluoride (FeF 3 ), iron(II) sulfate heptahydrate (FeSO 4 · 7 H 2 O), ammonium iron(II) sulfate hexahydrate (Fe(NH 4 ) 2 (SO 4 ) 2 · 6 H 2 O), iron chloride (FeC l2 ), iron(II) disulfide (FeS 2 ), iron(II) sulfate heptahydrate (FeSO 4 · 7 H 2 O), and iron(II) lactate hydrate (Fe(CH 3 CH(OH)COO) 2 -xH 2 O), L-iron(II) ascorbate (FeC 12 H 14 O 12 ) and iron(II) fluoride (FeF 2 ).

[0092] In a preferred embodiment, the iron excipient is ferrous sulfate hydrate (Fe 2 (SO 4 ) 3 ).

[0093] VII. Collagen It has been discovered by the present inventors that collagen powder and liquid collagen preparations can be utilized as bone graft materials. Type I collagen is a white, hygroscopic material that is commonly used as a component of bone grafts.

[0094] Collagen powder is a preparation of collagen material in which collagen polymers are cleaved chemically or enzymatically or by any conventional means to produce collagen fragments of a particular size or size range. In this preparation, the collagen can be 1 nm to 100 μm (microns) produced through reverse dialysis. In another preparation, the collagen can be enzymatically cleaved to produce fragments of 100 μm (microns) to 300 μm (microns). In yet another preparation, the collagen can be mechanically crushed or milled and then passed through a filter to produce fragments between 200 μm (microns) and 5 mm. In another embodiment, the collagen has a particle size of 25 μm (microns) to 750 μm (microns). In another embodiment, the collagen has a particle size of 1 μm (microns) to 1.5 mm (millimeters). In yet another preparation, the collagen is cryogenically crushed or milled (cryogrinding) to produce fragments of 1 μm (microns) to 5 mm.

[0095] Liquid collagen is a preparation of collagen that contains a soluble fraction of collagen dissolved in solution. In this preparation, collagen is soaked in an acid solution and / or heated to between 100°C and 300°C and / or heated in a pressurized environment and / or filtered to produce fragments of a specific size. In yet another preparation, liquid collagen is produced by grinding or milling collagen suspended in a solution with a rotor stator to produce collagen fragments between 1 μm (micron) and 1 mm.

[0096] In a preferred preparation, collagen is ground to produce a powder having one of the following size fragments: Fractions of 10 μm (microns), 15 μm (microns), 20 μm (microns), 25 μm (microns), 30 μm (microns), 35 μm (microns), 40 μm (microns), 50 μm (microns), 55 μm (microns), 60 μm (microns), 65 μm (microns), 70 μm (microns), 75 μm (microns), 80 μm (microns), 85 μm (microns), 90 μm (microns), 95 μm (microns), 100 μm (microns), 150 μm (microns), 200 μm (microns), 250 μm (microns), 300 μm (microns), 350 μm (microns), 400 μm (microns), 450 μm (microns), 500 μm (microns), 750 μm (microns), or 1000 μm (microns). In another preparation, the collagen powder is composed of fragment sizes in the above ranges.

[0097] VIII. Antibacterial Agents As used herein, the term "antimicrobial agent" refers to an agent that kills or stops the growth of microorganisms. Antibiotics are antibacterial agents that kill bacteria, and antifungals are agents that kill fungi.

[0098] Thus, in some embodiments, the bone graft composition comprises an antimicrobial agent, hi some embodiments, the antimicrobial agent comprises an antibiotic agent or an antifungal agent.

[0099] In some embodiments, the antibiotic comprises vancomycin, gentamicin, tobramycin, kanamycin, neomycin, ampicillin, methicillin, nafcillin, oxacillin, penicillin, ticarcillin, ciprofloxacin, vancomycin, cefazolin, cefepime, ceftriaxone, clindamycin, aztreonam, imipenem, quinupristin / dalfopristin, chloramphenicol, doxycycline, metronidazole, nitrofurantoin, polymyoxin B, tetracycline, viomycin, chloromycetin, streptomycin, azactam, pharmaceutically acceptable salts thereof, and combinations thereof.

[0100] In some embodiments, the antifungal agent comprises a polyene antifungal agent, an imidazole, a triazole, an allylamine, or an echinocandin. In some embodiments, the polyene antifungal agent comprises amphotericin B, candicidin, filipin, hamycin, natamycin, nystatin, rimocidin, an imidazole, a triazole, and a thiazole. In some embodiments, the imidazole comprises bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, and a triazole. In some embodiments, the triazoles include albaconazole, efinaconazole, epoxiconazole, fluconazole, isavuconazole, itraconazole, posaconazole, propiconazole, ravuconazole, terconazole, and voriconazole. In some embodiments, the allylamines include amorolfine, butenafine, naftifine, and terbinafine. In some embodiments, the echinocandins include anidulafungin, caspofungin, and micafungin.

[0101] In some embodiments, the bone graft composition exhibits antibacterial or antifungal efficacy.

[0102] IX. Dilute Solutions The diluent solution is an aqueous medium consisting of water and other excipients capable of solubilizing the powder or dry slurry components of the bone graft composite material.

[0103] In some embodiments, the bone graft composition includes a diluent or solution that is added to other components of the bone graft material to produce a malleable, moldable putty.

[0104] In some embodiments, the diluent is saline or water.

[0105] In a preferred embodiment, the diluent is 0.9% saline, as commonly used in some medical and scientific applications.

[0106] In another preferred embodiment, the diluent is 0.9% saline containing an acidifying agent.

[0107] In yet another preferred embodiment, the diluent is 0.9% saline containing acetic acid.

[0108] In yet another preferred embodiment, the diluent is 0.9% saline containing acetic acid and any acidifying agent including a citric acid molecule, e.g., citric acid monohydrate or magnesium citrate, potassium citrate, or sodium citrate dihydrate.

[0109] In another preferred embodiment, the diluent is 0.9% saline containing an acidifying agent and a liquid preparation of collagen.

[0110] X. Preparation of Bone Graft Composition The term "blending" as used herein refers to the physical process of combining or combining non-aqueous materials to form one substance or mass that is a powder or dry slurry, referred to herein as a "powder-dry slurry." Part of the preparation of a bone graft composite requires blending the following components into a single powder dry slurry composite: calcium phosphate with setting agent, iron excipient, agent to control setting rate, acidifying agent, and powdered collagen.

[0111] The term "mixing" as used herein refers to the physical process of combining a powdered dry slurry with a dilute solution to form a paste, putty, or other amorphous solid.

[0112] In some embodiments, 0.1 mL to 15 mL of diluent will be added to 0.1 g to 20 g of powder dry slurry. In some embodiments, 0.5 mL of diluent will be added to 0.5 g of powder dry slurry. In some embodiments, 0.765 mL of diluent will be added to 1 g of powder dry slurry. In some embodiments, 1 mL of diluent will be added to 1 g of powder dry slurry. In some embodiments, 1.5 mL of diluent will be added to 1.5 g of powder dry slurry. In some embodiments, 2 mL of diluent will be added to 2 g of powder dry slurry. In some embodiments, 2.5 mL of diluent will be added to 2.5 g of powder dry slurry. In some embodiments, 3 mL of diluent will be added to 3 g of powder dry slurry. In some embodiments, 3.5 mL of diluent will be added to 3.5 g of powder dry slurry. In some embodiments, 4 mL of diluent would be added to 4 g of powder dry slurry. In some embodiments, 4.5 mL of diluent would be added to 4.5 g of powder dry slurry. In some embodiments, 5 mL of diluent would be added to 5 g of powder dry slurry. In some embodiments, 5.5 mL of diluent would be added to 5.5 g of powder dry slurry. In some embodiments, 6 mL of diluent would be added to 6 g of powder dry slurry. In some embodiments, 6.5 mL of diluent would be added to 6.5 g of powder dry slurry. In some embodiments, 7 mL of diluent would be added to 7 g of powder dry slurry. In some embodiments, 7.5 mL of diluent would be added to 7.5 g of powder dry slurry. In some embodiments, 8 mL of diluent would be added to 8 g of powder dry slurry. In some embodiments, 8.5 mL of diluent would be added to 8.5 g of powder dry slurry. In some embodiments, 9 mL of diluent will be added to 9 g of powder dry slurry. In some embodiments, 9.5 mL of diluent will be added to 9.5 g of powder dry slurry.In some embodiments, 10 mL of diluent will be added to 10 g of powder dry slurry. In some embodiments, 10.5 mL of diluent will be added to 10.5 g of powder dry slurry.

[0113] In some embodiments, the bone graft composition has a density of 1.6 g / mL, 1.65 g / mL, 1.7 g / mL, 1.75 g / mL, 1.76 g / mL, 1.77 g / mL, 1.78 g / mL, 1.79 g / mL, 1.8 g / mL, 1.85 g / mL, 1.9 g / mL, or in the range of 1-6 g / mL after mixing. In some embodiments, the bone graft composition has a density of 2-6 g / mL after mixing. In some embodiments, the bone graft composition has a density of 2-5 g / mL after mixing. In some embodiments, the bone graft composition has a density of 3.33-4.2 g / mL after mixing.

[0114] In some embodiments, the force required to push the bone graft composition through the cannula, tube, or syringe is less than 100 N / m. In some embodiments, the force required to push the bone graft composition through the cannula, tube, or syringe is less than 90 N / m. In some embodiments, the force required to push the bone graft composition through the cannula, tube, or syringe is less than 80 N / m. In some embodiments, the force required to push the bone graft composition through the cannula, tube, or syringe is less than 70 N / m. In some embodiments, the force required to push the bone graft composition through the cannula, tube, or syringe is less than 60 N / m.

[0115] XI. Delivery of Active Agents A. Small molecule surfactants The inventors have discovered that bone graft materials can be designed to more efficiently solubilize, sequester and deliver small molecule active agents.

[0116] In some embodiments, the bone graft composition includes a small molecule active agent, which is a chemical species that affects some cellular function and is contained or sequestered in the bone graft composite material.

[0117] The small molecule active agent is a chemical species derived from a protein, a synthetic chemical species, or a chemical species derived from a plant or animal source. The chemical species can be chemically altered prior to incorporation into the bone graft composite material, or the chemical species can be in its native state prior to incorporation into the bone graft composite material.

[0118] The chemical properties, such as pH and osmolality, of the diluted solution components of the bone graft can be designed to increase the solubility of small molecule active agents in the diluted solution prior to mixing with the powder or dry slurry components of the bone graft composition.

[0119] The chemical properties of the powdered dry slurry components of the bone graft can be designed such that when the powdered dry slurry components are mixed with a diluent, the small molecule active agent is less likely to dissolve and is sequestered within the material, which can be exploited to control the amount of leaching and bioavailability of the small molecule active agent.

[0120] In some embodiments, the small molecule active agent may be a component of the diluent.

[0121] In some embodiments, the small molecule active agent can be a component of the powder dry slurry component.

[0122] In some embodiments, the small molecule active agent can be a separate preparation, either in liquid form or as a lyophilized solid, that is added to the diluent.

[0123] In some embodiments, the small molecule active agent can be a separate preparation, either a liquid or a lyophilized solid, that is added to the powdered dry slurry component.

[0124] In some embodiments, the small molecule is an opioid growth factor antagonist.

[0125] B. Other Biologically Derived Surfactants The present inventors have discovered that bone graft materials can be designed to more efficiently solubilize, sequester, and deliver biologically derived active agents.

[0126] In some embodiments, the bone graft composition includes a bioactive agent, which is a biological species that effects some cellular function and is contained or sequestered in the bone graft composite material.

[0127] The biologically derived active agents are chemical species derived from proteins, synthetically derived chemicals, or from plant or animal sources. The biologically derived species can be chemically altered prior to incorporation into the bone graft composite material, or the biologically derived species can be in its native state prior to incorporation into the bone graft composite material.

[0128] The chemical properties, such as pH and osmolality, of the diluted solution components of the bone graft can be designed to increase the solubility of the biologically active agent in the diluted solution prior to mixing with the powder or dry slurry components of the bone graft composition.

[0129] The chemical properties of the powdered dry slurry components of the bone graft can be designed such that when the powdered dry slurry components are mixed with a diluent, the bioactive agent is less likely to dissolve and is sequestered within the material, which can be used to control the amount of leaching and bioavailability of the bioactive agent.

[0130] In some embodiments, the bioactive agent may be a component of a dilute solution.

[0131] In some embodiments, the bioactive agent may be a component of the powder dry slurry component.

[0132] In some embodiments, the biologically active agent can be a separate preparation, either in liquid form or as a lyophilized solid, that is added to the diluent.

[0133] In some embodiments, the biologically derived active agent may be a separate preparation, either a liquid or a lyophilized solid, that is added to the powdered dry slurry component.

[0134] The additional bioactive agent may be a bone morphogenetic protein (i.e., BMP2, BMP4, BMP7), a platelet derived growth factor (PDGF) protein (i.e., PDGF-α or PDGF-β), a transforming growth factor (TGF) β protein, a vascular endothelial growth factor (VEGF) protein, cyclopamine, purmorphamine, resolfam, a netrin protein (i.e., netrin-1 or netrin-4), a slit protein (i.e., slit-1, slit-2, slit-3), a repulsive guidance molecule (RGM) protein, or a WNT protein (i.e., WNT1, WNT5, WNT10, WNT11).

[0135] C. Opioid Growth Factor Receptor (OGFR) Antagonists By "OGFR antagonist" is meant any small molecule active agent that inhibits, suppresses, or abolishes at least one OGFR-associated biological activity.

[0136] In some embodiments, the OGFR antagonist is an OGFR binding antagonist, ie, a molecule that interferes with, blocks, or otherwise prevents the interaction or binding of met5-ligand (OGF) to OGFR.

[0137] The OGFR binding antagonist can function in two ways. First, the OGFR antagonist can compete with the met5-ligand for binding to the OGFR on the nuclear membrane surface, thereby interfering with, blocking or otherwise preventing the binding of the met5-ligand to the OGFR, and bind to the OGFR without triggering downstream signaling induced by the binding of the met5-ligand to the OGFR. Alternatively, the OGFR binding antagonist can bind to or sequester PENK or the met5-ligand with sufficient affinity and specificity to substantially interfering with, blocking or otherwise preventing the binding of the met5-ligand to the OGFR, thereby inhibiting, suppressing or abolishing at least one OGFR-related biological activity. In general, the OGFR binding antagonist can be a large molecule (e.g., an antibody) or a small molecule (e.g., a compound with a molecular weight of less than 15 kD, 12 kD, 10 kD or even 8 kD), and can be a polypeptide, a nucleic acid, or a synthetic small molecule compound. OGFR binding antagonist can be identified by any in vitro assay that is easily selected by those skilled in the art.For example, OGFR antagonist can be identified by using the method described in U.S. Patent No. 5,882,944, U.S. Patent No. 6,007,986, U.S. Patent No. 6,270,979.

[0138] In one embodiment, the OGFR binding antagonist is naloxone or a functional derivative thereof, naltrexone or a functional derivative thereof, or a combination thereof.

[0139] As used herein, "functional derivative" refers to a derivative or analogue that is structurally and functionally similar to the starting molecule (e.g., maintains the function of naltrexone or naloxone as an OGFR antagonist). Naloxone and naltrexone analogues can be synthesized using standard synthetic procedures, such as those described in March J., Advanced Organic Chemistry, 3rd Ed. (1985). Examples of naltrexone and naloxone functional derivatives include salt forms, such as naloxone hydrochloride dihydrate or naltrexone hydrochloride. Additional examples of naltrexone and naloxone functional derivatives suitable for use in the methods of the present invention include naltrexone and naloxone analogues, such as those described in U.S. Patent Application Publication No. 2007 / 0197573 A1, U.S. Patent No. 6,713,488.

[0140] In another embodiment, the OGFR binding antagonist is derived from oxymorphone and binds to OGFR, including naloxone, naltrexone, nalorphine, naloxonazine, levallorphan, nalmefene, cyprodim, cilorphan, cyclazocine, oxilorphan, LY113878, MR2266, diprenorphine, WIN 44,441-3, naltoindole, or norbinaltorphimine.

[0141] In yet another embodiment, the OGFR binding antagonist is derived from trans-3,4-dimethyl-4-phenylpiperidine and binds to OGFR, including LY99335, LY25506, LY117413, or LY255582.

[0142] In another embodiment, the OGFR binding antagonist is derived from a met5-enkephalin or Leu-enkephalin peptide and contains, at a minimum, the following amino acid sequences as a means of binding to and targeting the OGFR: Tyr-Gly-Gly-Phe-Met (SEQ ID NO: 1) for those derived from met5-enkephalin, or Tyr-Gly-Gly-Phe-Leu (SEQ ID NO: 2) for those derived from Leu-enkephalin.

[0143] In yet another embodiment, the OGFR binding antagonist is derived from the peptide antagonist 101174864 (N,N-Diallyl-Tyr-Aib-Aib-Phe-Leu-OH, SEQ ID NO: 3; Aib = aminoisobutyric acid) or the somatostatin analog CTP (D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH.sub.2, SEQ ID NO: 4).

[0144] In other embodiments, the OGFR antagonist is, instead of being an OGFR binding antagonist, a molecule that inhibits the nuclear localization sequence found within OGFR: 251QSALDYFMFAVRCRHQRRQLVHFAWEHFRPRCKFVWGPQDKLRRFKPSSL (SEQ ID NO:5).

[0145] In yet another embodiment, the OGFR antagonist employed in the present methods is a small hairpin (sh)-RNA or a small interfering (si)-RNA directed against the OGFR gene and effective to inhibit OGFR gene expression.

[0146] The OGFR antagonists described herein can be administered individually or in combination. Suitable combinations include, for example, naloxone and naltrexone; naloxone and / or naltrexone in combination with another OGFR binding antagonist or another OGFR antagonist.

[0147] D. Topical Delivery Systems and Carriers In one embodiment of the present disclosure, the bone graft composite material is manufactured by mixing a suitable diluent containing the active agent with a powder dry slurry that is administered directly and locally to the site of a bone injury or surgical procedure where the bone graft material would normally be placed to stimulate bone formation.

[0148] The inventors have discovered that specific formulations of the diluent and powder dry slurry components can be achieved to optimize the dissolution, sequestration, and bioavailability of OGFR antagonists that share chemical properties with either naloxone, naltrexone, a combination of naloxone and naltrexone, or salts thereof.

[0149] In some embodiments, the OGFR antagonist is incorporated into the bone graft composite material such that the concentration in the material is between 1 nanomole (nM) per cubic centimeter (1 nM / cc) and about 100 mM per cubic centimeter (100 mm / cc). In some embodiments, the concentrations are 1 mM / cc, 1.25 mM / cc, 1.5 mM / cc, 1.75 mM / cc, 2.0 mM / cc, 3.0 mM / cc, 4.0 mM / cc, 5.0 mM / cc, 6.0 mM / cc, 7.0 mM / cc, 8.0 mM / cc, 9.0 mM / cc, and / or 10.0 mM / cc, 20.0 mM / cc, 30.0 mM / cc, 40.0 mM / cc, 50.0 mM / cc, 60.0 mM / cc, 70.0 mM / cc, 80.0 mM / cc, 90.0 mM / cc, 100 mM / cc.

[0150] In some embodiments, the concentration of the OGFR antagonist in the bone graft material is such that the elution rate from the material is between 0.001 pM per second and 10 mM per day.

[0151] In some embodiments, the cumulative dose resulting from elution of the OGFR agonist during the period in which the small molecule is released from the bone graft material by diffusion or other physical effects resulting from the normal degradation of the bone graft material once implanted is any of 0.8 mM, 1 mM, 1.1 mM, 1.15 mM, 1.2 mM, 1.25 mM, 1.3 mM, 1.35 mM, 1.4 mM, 1.45 mM, 1.5 mM, 1.55 mM, 1.6 mM, 1.65 mM, 1.7 mM, 1.75 mM, 1.8 mM, 1.9 mM, 2 mM, 2.7 mM, 3 mM, 3.6 mM, 4 mM, 4.5 mM, 5 mM, 5.4 mM, 6 mM, 6.3 mM, 7 mM, 7.2 mM, 8 mM, 8.1 mM, 9 mM or 10 mM.

[0152] In a preferred embodiment, the OGFR antagonist is naloxone hydrochloride at a concentration of 0.2 mg / cc (0.5 mM / cc), 0.25 mg / cc (0.625 mM / cc), 0.3 mg / cc (0.75 mM / cc), 0.35 mg / cc (0.875 mM / cc), 0.4 mg / cc (1 mM / cc), 0.45 mg / cc (1.125 mM / cc), 0.5 mg / cc (1.25 mM / cc), 0.55 mg / cc (1.37 mM / cc), 0.5 mg / cc (1.4 mM / cc), 0.65 mg / cc (1.8 mM / cc), 0.75 mg / cc (1.6 mM / cc), 0.85 mg / cc (1.8 mM / cc), 0.95 mg / cc (1.8 mM / cc), 0.95 mg / cc (1.6 mM / cc), 0.95 mg / cc (1.8 ... 5mM / cc), 0.6mg / cc(1.5mM / cc), 0.65mg / cc(1.625mM / cc), 0.7mg / cc(1.75mM / cc), 0.75mg / cc(1.875mM / cc), 0.8mg / cc (2mM / cc), 0.85mg / cc (2.125mM / cc), 0.9mg / cc (2.25mM / cc), 0.95mg / cc (2.375mM / cc) or 1mg / cc (2.5mM / cc).

[0153] E. Composition and Administration The methods and compositions herein may be provided in the form of a kit. A "kit" is defined herein as a package containing several individual components that exhibit complementary effects when applied together. In this embodiment, the effects achieved by the kit and the pharmaceutical composition are similar. The kit may optionally include instructions for using the pharmaceutical composition.

[0154] The present invention is further illustrated by the following non-limiting examples.

[0155] Example 1: Bone graft material

[0156] In a first embodiment, a preferred embodiment of the dry slurry components of the bone graft material includes the following as weight percentages: 1) Iron(III) chloride between 1% and 7% 2) Calcium phosphate dibasic between 8.5% and 11% 3) 9.5% to 12.5% ​​calcium phosphate tribasic 4) 15% to 17% hydroxyapatite 5) 3% to 6% β-tricalcium phosphate 6) 5% to 7.5% sodium phosphate dibasic 7) 12% to 16% sodium carbonate 8) 2% to 6% calcium oxide 9) 4% to 6% magnesium oxide 10) 0.5% to 2.5% L-ascorbic acid and 11) 19% to 23% powdered type I collagen. A preferred embodiment of the diluent component is a solution having a volume of 0.5 mL to 12.5 mL consisting of: 1) 0.9% saline 2) 2% to 6% acetic acid 3) 0.1% to 2% citric acid monohydrate and 4) 0.01% to 2% naloxone hydrochloride. Upon mixing the dry slurry components and diluent, the bone graft material becomes moldable after 3 minutes and solidifies to a light brown color, reaching a peak temperature of 29.7°C 2 minutes after mixing.

[0157] In a second embodiment, a preferred embodiment of the dry slurry components of the bone graft material includes the following as weight percentages: 1) 5% to 10% iron(III) citrate 2) 8% to 11% dibasic calcium phosphate 3) 9% to 12.5% ​​calcium phosphate tribasic 4) 13% to 17% hydroxyapatite 5) 3% to 6% β-tricalcium phosphate 6) 4% to 7.5% sodium phosphate dibasic 7) 11% to 15% sodium carbonate 8) 2% to 6% calcium oxide 9) 4% to 6% magnesium oxide 10) 0.5% to 2.5% L-ascorbic acid and 11) 19% to 23% powdered type I collagen. A preferred embodiment of the diluent component is a solution having a volume between 0.5 mL and 12.5 mL, comprising: 1) 0.9% saline 2) 2% to 6% acetic acid 3) 0.1% to 2% citric acid monohydrate and 4) 0.01% to 2% naloxone hydrochloride. Upon mixing the dry slurry components and diluent, the bone graft material becomes moldable after 5 minutes and solidifies to a dark chocolate brown color, reaching a peak temperature of 28.6° C. 30 seconds after mixing.

[0158] In a third embodiment, a preferred embodiment of the dry slurry components of the bone graft material includes the following as weight percentages: 1) 1% to 5% iron(III) oxide 2) 8% to 11% dibasic calcium phosphate 3) 9% to 13% calcium phosphate tribasic 4) 14% to 18% hydroxyapatite 5) 3% to 6.5% β-tricalcium phosphate 6) 4% to 8% sodium phosphate dibasic 7) 12% to 16% sodium carbonate 8) 2% to 6% calcium oxide 9) 4% to 6% magnesium oxide 10) 0.5% to 2.5% L-ascorbic acid and 11) 20% to 24% powdered type I collagen. A preferred embodiment of the diluent component is a solution having a volume between 0.5 mL and 12.5 mL, comprising: 1) 0.9% saline 2) 2% to 6% acetic acid 3) 0.1% to 2% citric acid monohydrate and 4) 0.01% to 2% naloxone hydrochloride Upon mixing of the dry slurry components and diluent, the bone graft material was moldable after 3.5 minutes, solidified to a white color with iron gray tint, and reached a peak temperature of 29.8°C 30 seconds after mixing.

[0159] In a fourth embodiment, a preferred embodiment of the dry slurry components of the bone graft material includes the following as weight percentages: 1) 5% to 10% iron(III) phosphate 2) 8% to 11% dibasic calcium phosphate 3) 9% to 12.5% ​​calcium phosphate tribasic 4) 13% to 17% hydroxyapatite 5) 3% to 6% β-tricalcium phosphate 6) 4% to 7.5% sodium phosphate dibasic 7) 12% to 16% sodium carbonate 8) 2% to 6% calcium oxide 9) 4% to 6% magnesium oxide 10) 0.5% to 2.5% L-ascorbic acid and 11) 19% to 23% powdered type I collagen. A preferred embodiment of the diluent component is a solution having a volume between 0.5 mL and 12.5 mL, comprising: 1) 0.9% saline 2) 2% to 6% acetic acid 3) 0.1% to 2% citric acid monohydrate and 4) 0.01% to 2% naloxone hydrochloride. Upon mixing the dry slurry components and diluent, the bone graft material becomes moldable after 3.5 minutes and solidifies to a light brown color, reaching a peak temperature of 33.5° C. 1.5 minutes after mixing.

[0160] In a fifth embodiment, a preferred embodiment of the dry slurry components of the bone graft material includes the following as weight percentages: 1) 15% to 10% ammonium iron(III) citrate 2) 8% to 11% dibasic calcium phosphate 3) 9% to 12.5% ​​calcium phosphate tribasic 4) 13% to 17% hydroxyapatite 5) 3% to 6% β-tricalcium phosphate 6) 4% to 7.5% sodium phosphate dibasic 7) 11% to 15% sodium carbonate 8) 2% to 6% calcium oxide 9) 4% to 6% magnesium oxide 10) 0.5% to 2.5% L-ascorbic acid and 11) 19% to 23% powdered type I collagen. A preferred embodiment of the diluent component is a solution having a volume between 0.5 mL and 12.5 mL, comprising: 1) 0.9% saline 2) 2% to 6% acetic acid 3) 0.1% to 2% citric acid monohydrate and 4) 0.01% to 2% naloxane hydrochloride. Upon mixing the dry slurry components and diluent, the bone graft material becomes moldable after 3 minutes and turns brown as it solidifies, reaching a peak temperature of 30.3° C. 1.5 minutes after mixing.

[0161] In a sixth embodiment, a preferred embodiment of the dry slurry components of the bone graft material includes the following as weight percentages: 1) Iron(III) sulfate between 5% and 12% 2) 8% to 11% dibasic calcium phosphate 3) 9% to 12.5% ​​calcium phosphate tribasic 4) 13% to 17% hydroxyapatite 5) 3% to 6% β-tricalcium phosphate 6) 4% to 7.5% sodium phosphate dibasic 7) 11% to 15% sodium carbonate 8) 2% to 6% calcium oxide 9) 4% to 6% magnesium oxide 10) 0.5% to 2.5% L-ascorbic acid and 11) powdered type I collagen, 18% or more and 23% or less. A preferred embodiment of the diluent component is a solution having a volume between 0.5 mL and 12.5 mL, comprising: 1) 0.9% saline 2) 2% to 6% acetic acid and 3) 0.1% to 2% citric acid monohydrate. In this embodiment, no active agent is incorporated. Upon mixing the dry slurry components and diluent, the bone graft material becomes moldable after approximately 2.5 minutes and solidifies to a dark brown color, reaching a peak temperature of 38° C. 1.5 minutes after mixing.

[0162] In a seventh embodiment, a preferred embodiment of the dry slurry components of the bone graft material includes the following as weight percentages: 1) Iron(III) sulfate between 5% and 12% 2) 8% to 11% dibasic calcium phosphate 3) 9% to 12.5% ​​calcium phosphate tribasic 4) 13% to 17% hydroxyapatite 5) 3% to 6% β-tricalcium phosphate 6) 4% to 7.5% sodium phosphate dibasic 7) 11% to 15% sodium carbonate 8) 2% to 6% calcium oxide 9) 4% to 6% magnesium oxide 10) 0.5% to 2.5% L-ascorbic acid and 11) 18% to 23% powdered type I collagen. A preferred embodiment of the diluent component is a solution having a volume between 0.5 mL and 12.5 mL, comprising: 1) 0.9% saline 2) 2% to 6% acetic acid 3) 0.1% to 2% citric acid monohydrate and 4) 0.01% to 2% naloxone hydrochloride. The dry slurry ingredients and diluent are mixed together and become moldable in approximately 2.5 minutes, solidify to a dark brown color, and reach a peak temperature of 38° C. 1.5 minutes after mixing.

[0163] Additionally, in a seventh preferred embodiment containing ferrous sulfate, bone graft material was mixed as described and placed in culture to assess osteoblast differentiation according to ASTM F3106-14. MC3T3-E1 (MC3) cells, a mouse osteoblast (bone cell) precursor cell line, were seeded in culture as micromasses (2.5 x 105 cells per micromass suspended in 20 μL of Matrigel + DMEM solution), grown for 120 hours, and maintained in growth medium (DMEM with 20% fetal bovine serum). Control cultures (Control) were maintained in growth medium. Induced control cultures (Induced) were stimulated to become osteocytes with medium supplemented with ascorbic acid, β-glycerophosphate, and dexamethasone. In this example, MC3 micromass spheroids were grown for an additional 7 days, after which experimental groups were stained with Alizarin Red and analyzed for mineral content using one-way ANOVA with Bonferroni correction to assess differences.

[0164] The results demonstrate that cultures containing ferrous sulfate material (with or without activator) significantly increased bone formation based on Alizarin Red staining (Figure 1A) compared to induced and control cultures. The size of the micromass spheroids and the mineral contained in each micromass spheroid were quantified to identify the mineral area fraction. The mineral area fraction of micromass spheroids with ferrous sulfate bone graft material had significantly more mineral by 4-fold (*=p<0.0046) than the control cultures (Figure 1B). From these data, it can be concluded that the ferrous sulfate bone graft material is osteoconductive and osteopromoting. In between-group analysis, bone formation in bone grafts without naloxone demonstrated the components of the bone graft material that increased mineral formation by 4-fold (*=p<0.0046) in cultures versus control and wells chemically induced to make bone. The addition of 0.4 mg of naloxone to the bone graft material increased mineral formation by an additional 25% (x = p<0.0001) versus the control bone graft material group without naloxone.

[0165] Example 2: Use of bone graft implants in spinal fusion surgery

[0166] Example 2 shows lateral lumbar interbody fusion (LLIF) using the claimed bone graft. In particular, in this study, LIF is performed using an osteoconductive synthetic bone graft material containing low molecular weight naloxone hydrochloride. The synthetic bone graft is composed of bovine type I collagen, hydroxyapatite (HA), β-tricalcium phosphate, and other calcium salts and some excipients. Hereinafter, the synthetic bone graft composition containing naloxone hydrochloride is referred to as composition ZF.

[0167] The present disclosure provides a method of repairing a bone defect in a patient in need thereof, comprising applying a bone graft composition, such as composition ZF, which includes a surgical procedure, such as a lumbar interbody fusion procedure (LIF). In some embodiments, the LIF procedure includes anterior LIF (ALIF), lateral LIF (LLIF), transforaminal LIF (TLIF), and posterior LIF (PLIF). In some embodiments, the LIF procedure alleviates an associated pathology, such as degenerative disc disease in a subject, as determined through radiography, by the formation of a bone defect through a surgical procedure. In some embodiments, the LIF procedure alleviates an associated neuromuscular disorder or physical defect in a subject, as determined through the subject's Oswestry Disability Index (ODI), through a neurological evaluation of the subject, or through radiography of the subject.

[0168] Background of LIF treatment

[0169] LIF procedures are differentiated based on the anatomical approach and hardware implanted and include anterior LIF (ALIF), lateral LIF (LLIF), transforaminal LIF (TLIF), and posterior LIF (PLIF). LIF procedures involve removing the disc believed to be causing low back or leg pain and replacing it with an interbody cage or spacer to promote disc-vertebral fusion. LIF procedures have increased significantly over the past two decades due to evolution of surgical techniques, implants, imaging modalities, and demands.

[0170] As a result of the potential challenges of local autografts, allografts and orthobiologics are routinely used in combination with or as a substitute for local bone to meet the needs of bone grafting in LIF procedures. Although great strides have been made in the development of bone graft materials, there remains a need for bone graft materials that provide biomechanically strong, rigid fixation and have a good safety profile.

[0171] Testing of bone graft compositions

[0172] Indications for synthetic ZF bone graft include spinal fusion in skeletally mature patients with degenerative disc disease (DDD) at one level, L2-L5. DDD is defined as discogenic back pain with disc degeneration and / or grade 2 spondylolisthesis at the involved level, confirmed by patient history and radiographic evaluation. Patients receiving synthetic ZF bone graft have failed non-operative treatment for at least 3 months prior to treatment with the synthetic ZF bone graft device. In lateral lumbar interbody fusion (LLIF), synthetic ZF bone graft is implanted with a commercially available polyetheretherketone (PEEK), porous PEEK, or hydroxyapatite (HA) PEEK spacer and ancillary fixation system (lateral plate or bilateral pedicle screws).

[0173] Study design

[0174] Subjects will undergo LLIF with auxiliary fixation system or auxiliary fixation consisting of lateral plate. In the experimental group, synthetic ZF bone graft will be filled into the cavity of the intervertebral body spacer. In the control group, allogeneic bone graft (i.e., DBX putty, demineralized bone matrix) + / - bone marrow fluid will be filled into the cavity of the intervertebral body spacer. The eligibility criteria for surgery are as follows: 1) Patients aged 22 to 75 years. 2) Diagnosis of degenerative disc disease (DDD) associated with back pain, with or without leg pain, at a single level of the spine between the second lumbar vertebra (L2) and the fifth lumbar vertebra (L5), confirmed by medical history and radiographic evaluation (e.g., plain radiograph, computed tomography (CT), magnetic resonance imaging (MRI)). 3) Spinal instability as evidenced by x-rays taken during flexion and extension showing vertebral angle of 5 degrees or more and / or translation of 4 mm or more in the affected area. 4) Imaging pathology showing osteophyte formation, loss of disc height, ligament thickening, disc degeneration / herniation, facet joint degeneration, dark disc appearance on MRI, foraminal narrowing, or loss of foraminal height. 5) DDD is defined as discogenic low back pain with evidence of degenerative disc disease documented by medical history and radiological examination, and patients with DDD may also have spondylolisthesis up to grade 1 at the involved spinal level. 6) Patients who had failed non-operative treatment for at least 6 months prior to surgery. 7) Lateral lumbar interbody fusion (LIF) uses a PEEK or titanium spacer and an adjunctive fixation system (lateral plate or bilateral pedicle screws).

[0175] Example 3: Use of bone void filler for bone voids or gaps that are not essential to the stability of the bone structure.

[0176] Uses of the bone graft compositions disclosed herein include using the bone graft compositions as bone void fillers for bone voids or gaps that are not essential to the stability of bone structures. In particular, synthetic ZB bone graft (a composition identical to synthetic ZF except that it lacks naloxone) is gently filled into bone voids or gaps in the skeletal system (e.g., the posterolateral spine, pelvis, ilium, and / or extremities).

[0177] Thus, the present disclosure provides a method of repairing a bone defect in a patient in need thereof, comprising applying a bone graft composition disclosed herein to the bone defect, wherein the bone defect comprises a void or gap in the bony skeletal system, where the void or gap is not essential to the stability of the bony skeletal structure, and the bone graft composition fills the void or gap. In some embodiments, the defects are surgically created bony defects or bony defects resulting from traumatic injury to the bone. The synthetic ZB bone graft resorbs and replaces bone during healing of the defect.

[0178] Example 4: Safety and Efficacy of Synthetic ZF Bone Grafts in Subjects Undergoing Reconstructive Procedures to Stabilize the Humerus Following Resection of Metastatic Breast Cancer Tumors

[0179] This study evaluates the safety and efficacy of synthetic ZF bone grafts using intercalary allografts and plate fixation in humeral shaft-end physeal reconstruction after resection of malignant tumors, including metastatic breast cancer.

[0180] Study design

[0181] Subjects in the experimental group will receive a synthetic ZF bone graft containing an osteoconductive synthetic bone graft material and the small molecule naloxone hydrochloride. The synthetic bone graft is composed of bovine type I collagen, hydroxyapatite (HA), beta-tricalcium phosphate, and other calcium salts and several excipients. Subjects in the control group will receive an allograft fixed with plates and screws, which will be filled with polymethylmethacrylate (PMMA) bone cement. Establishing non-inferiority of the experimental group to the control group will validate the validity of this study.

[0182] Example 5: Safety and Efficacy of Synthetic ZF Bone Grafts in Subjects Undergoing Reconstructive Procedures to Stabilize the Femur, Tibia, or Pelvis Following Resection of Metastatic Breast Cancer Tumors

[0183] This study evaluates the safety and efficacy of synthetic ZF bone grafts in reconstructing the proximal femur, distal femur, tibia, and pelvis using intramedullary nails, intramedullary locking nails, cephalomedullary nails, plate fixation, or a combination of orthopedic fixation devices after resection of malignant tumors, including metastatic breast cancer. After tumor resection, polymethylmethacrylate (PMMA) bone cement is placed into the defect to provide mechanical stability, and the synthetic ZF is placed around the implant / PMMA bone cement construct adjacent to the bone tissue.

[0184] Study design

[0185] The subjects in the experimental group will receive a suitable fixation device with PMMA bone cement and a synthetic ZF bone graft containing osteoconductive synthetic bone graft material and low molecular weight naloxone hydrochloride after tumor removal. The synthetic bone graft is composed of bovine type I collagen, hydroxyapatite (HA), β-tricalcium phosphate, other calcium salts and some excipients. The subjects in the control group will receive a suitable fixation device and PMMA bone cement after tumor removal. The establishment of non-inferiority of the experimental group to the control group confirms the validity of this study.

Claims

1. 1. A bone graft composition for repairing a bone defect in a patient in need of repair of the bone defect, comprising: The present invention comprises a calcium phosphate putty, an acidifying agent, collagen, an iron excipient, and a diluent solution, the iron excipient is selected from the group consisting of ferrous sulfate hydrate, ferrous chloride, ferrous citrate, ferrous oxide, ferrous oxide hydroxide, ferrous phosphate, ammonium ferrous citrate, elemental iron granules, ferrous fluoride, ferrous sulfate heptahydrate, ammonium ferrous sulfate hexahydrate, ferrous chloride, ferrous disulfide, ferrous lactate hydrate, ferrous L-ascorbate, and ferrous fluoride; The bone graft composition, wherein (i) the iron excipient is chromogenic, such that visual assessment of color uniformity during mixing of solid and liquid precursors is performed by observing the color due to the chromogenicity of the iron excipient, and (ii) the iron excipient acts as a radiographic contrast agent.

2. 10. The bone graft composition of claim 1, wherein the bone defect is caused by a human disease or condition, and the bone graft composition does not contain an active agent for treating the human disease or condition.

3. The bone graft composition of claim 2, wherein the human disease or condition is a bone void or defect that is not essential to the stability of bone structure, the bone void or defect being caused by surgery or trauma, and the bone graft composition fills the bone void or defect.

4. The bone graft composition of claim 1 , wherein the calcium phosphate putty comprises biphasic calcium phosphate particles.

5. The bone graft composition of claim 4 , wherein the biphasic calcium phosphate particles comprise hydroxyapatite and tricalcium phosphate.

6. The bone graft composition of claim 4, wherein the biphasic calcium phosphate particles comprise about 20-60% hydroxyapatite and about 40-80% tricalcium phosphate.

7. 2. The bone graft composition of claim 1, wherein the acidifying agent comprises one or more selected from the group of ascorbic acid, magnesium citrate, potassium citrate, sodium citrate, citric acid monohydrate, and acetic acid.

8. The bone graft composition of claim 1 , wherein the bone graft composition further comprises a hardening agent.

9. The bone graft composition of claim 8 , wherein the bone graft composition further comprises a hardening rate control agent.

10. 2. The bone graft composition of claim 1, wherein the iron excipient is ferrous sulfate hydrate.

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