BACKGROUND OF THE INVENTION

Diabetes mellitus (DM) is a chronic metabolic disease with high blood glucose levels.

Diabetes results from deficits in the production of insulin or deficit insulin resistance coupled with insufficient insulin production. Type 1 diabetes mellitus (T1DM) is due to the lack of insulin production by the pancreas and requires daily administration of insulin. It is typically caused by destruction of pancreatic β-cells of autoimmune etiology. Type 2 diabetes mellitus (T2DM) is characterized by the inability to use insulin efficiently, referred to as insulin resistance combined with an inability to produce a sufficient amount of insulin to overcome the insulin resistance. Diabetes mellitus often leads to serious complications that affect the heart, blood vessels, eyes, kidneys, and nerves. It has also been increasingly recognized that diabetes adversely affects bone health.

High levels of glucose contribute to diabetic complications by inducing stress at the cellular level, glycating proteins that lead to the formation of advanced glycation endproducts, increasing production of reactive oxygen species, and enhancing expression of cytokines such as tumor necrosis factor. In diabetic humans and animals there is increased production of inflammatory mediators by macrophages in adipose tissue leading to increased systemic inflammation, which among other factors contributes to insulin resistance. Diabetic conditions such as high glucose levels, increased formation of advanced glycation endproducts and increased generation of ROS lead to greater expression of inflammatory cytokines at the local level when tissues are perturbated by events such as wounding.

Pro-inflammatory mediators including TNF-α, IL-1β, IL-6 and IL-18 are increased locally in diabetes mellitus and are thought to contribute to diabetic complications. Diabetics have difficulty in down regulating inflammation once induced. Increased levels of TNF may limit the capacity of diabetics to down regulate other inflammatory genes and increase apoptosis, which has been shown to reduce bone coupling in diabetic animals.

During perturbation diabetes increases and prolongs inflammation, which may lead to enhanced osteoclastogenesis. Diabetes increases osteoclast formation in a number of conditions including periodontal disease, fracture healing and osteoporosis. Diabetes-increased osteoclasts may pertain to situations where bone is challenged by injury or inflammation rather than basal levels. Diabetes leads to increased RANKL/OPG ratios and TNF levels that contribute to greater bone resorption. In humans, the ratio of RANKL/OPG and TNF levels are increased in poorly controlled diabetics. Fatty acid levels in diabetics may also contribute to increased osteoclastogenesis. The capacity to resolve inflammation is an important aspect of limiting bone resorption as shown by diminished bone loss in animals treated with resolvins or by use of TNF inhibitors.

Diabetic fracture is a significant co-morbidity of both type I and type II diabetes and is characterized by microarchitectural changes that decrease bone quality. Meta-analysis shows a consistent pattern of increased risk of fracture in men and women and in studies conducted in the United States and Europe. For example, the relative risk of hip fracture is increased 6-7 fold for individuals with T1DM, which is considerably higher than the increased risk (1.4-1.7 fold) in T2DM. The fracture risk of T1DM increases because of a decrease of bone mineral density (BMD), which is linked to impaired bone formation that may be linked to a deficiency of insulin and insulin-like growth factor-1 (IGF-1). T2DM is often characterized by normal or high BMD. Diabetes may be associated with a reduction of bone strength that is not reflected in the measurement of BMD, which results in high risk of fracture.

Healing of fractures in diabetic patients is prolonged by 87% and has a 3.4 fold higher risk of complications including delayed union, non-union, redislocation or pseudoarthrosis. Clinical studies in humans indicate that diabetes delays fracture healing. A study of spontaneously diabetic animals revealed that diabetic fracture healing was characterized by decreased bone apposition and mineralization. The reparative phase of bone fracture healing is initiated by proliferation and chondroblastic differentiation of periosteal precursor cells resulting in a hyaline cartilage callus around the wounded bone. Imbalances in chondrocyte apoptosis, premature removal of cartilage, reduced osteoblast differentiation and function and alterations in vascularization have been shown to affect the transition from cartilage to bone. Supernormal osteoclast activity disturbs remodeling of the osseous callus. It has been proposed in the art that any of insulin insufficiency, hyperglycemia and oxidative stress are mechanisms that affect fracture healing in T1DM and T2DM. They may reduce osteoblast differentiation, increase osteoclast activity, and alter apoptosis of chondrocytes and osteoblasts to interfere with fracture healing in diabetic patients.

T1DM and T2DM both increase fracture risk and have several common mechanisms including increased AGE formation, increased ROS generation, and increased inflammation. These factors affect osteoblasts and osteoclasts. Both humans and animal models of T1DM and T2DM display impaired fracture healing although T1DM patients have a greater risk of developing fractures. Moreover, animals with T1DM and T2DM exhibit impaired bone formation under conditions of perturbation such as bacteria induced periodontal bone loss and bone fracture healing.

Developing biologically based methods for restoring skeletal healing deficiencies related to diabetes is of great clinical interest.

SUMMARY OF THE INVENTION

A specific signaling deficiency underlying poor fracture healing in the diabetic skeletal stem cell niche is identified, and demonstrated to be reversed by exogenous delivery of an agent that provides for Hedgehog activity, leading to restoration of normal levels of bone regeneration. It is shown herein that agents that provide hedgehog activity for this purpose may include, without limitation, native or modified hedgehog proteins; antibodies that bind to and activate a hedgehog pathway protein, e.g. smoothened, patched, etc.; small molecules that are agonists of smoothened or patched; and the like. Specific skeletal stem cell niche-related abnormalities that impair skeletal repair are present in diabetic patients. This deficiency can be reversed by targeted delivery of a hedgehog agent to the fracture site, for example using a sustained release formulation such as a hydrogel. In the presence of exogenously applied hedgehog activity the injury-induced expansion and osteogenic potential of skeletal stem cells is restored, culminating in the rescue of diabetic bone healing. The methods provided herein can correct deficient skeletal healing in diabetic patients for a variety of applications, from repair of skeletal fractures, to securing bone or teeth implants.

In one embodiment a method is provided for promoting bone healing in a diabetic patient in need thereof by locally administering a therapeutically effective amount of hedgehog agent to the patient. Another aspect relates to the use of a hedgehog agent in the manufacture of a medicament for accelerating bone healing in a diabetic patient in need thereof, which medicament is characterized by comprising a therapeutically effective amount of hedgehog agent for localized administration. An additional aspect provides a drug delivery device, which comprises a hedgehog agent and a pharmaceutically acceptable carrier, wherein the device is adapted for localized administration of the agent to a patient in need thereof.

In some embodiments the hedgehog agent is a hedgehog protein. The hedgehog protein may be a human protein, or a variant or active fragment thereof. In some embodiments the hedgehog protein is sonic hedgehog. In some embodiments the hedgehog protein is indian hedgehog. In other embodiments the hedgehog agent is a small molecule agonist.

In one embodiment, the patient is afflicted with a bone condition selected from bone fracture, bone trauma, arthrodesis, and a bone deficit condition associated with post-traumatic bone surgery, post-prosthetic joint surgery, post-plastic bone surgery, post-dental surgery, bone chemotherapy treatment, congenital bone loss, post traumatic bone loss, post surgical bone loss, post infectious bone loss, allograft incorporation or bone radiotherapy treatment.

In an embodiment, the hedgehog agent is delivered via an hedgehog agent delivery system. In one embodiment, the hedgehog agent delivery system includes at least one biocompatible carrier or matrix. In another embodiment, the biocompatible carrier includes poly-lactic acid, poly-glycolic acid, copolymers of poly-lactic acid or poly-glycolic acid. In yet another embodiment, the biocompatible carrier includes at least one bioerodible fatty acid or a metal salt of a fatty acid. In one embodiment the biocompatible carrier is a hydrogel, for example a poly(ethylene glycol) hydrogel. In yet another embodiment, the hedgehog agent delivery system includes an article for implantation. In an additional embodiment, the hedgehog agent delivery system includes a coating on an article for implantation.

In another embodiment, the carrier comprises porous or non-porous calcium phosphate, porous or non-porous hydroxyapatite, porous or non-porous tricalcium phosphate, porous or non-porous tetracalcium phosphate, porous or non-porous calcium sulfate, or a combination thereof. The delivery system may further comprise a bioactive bone agent, including without limitation peptide growth factors, anti-inflammatory factors, pro-inflammatory factors, inhibitors of apoptosis, MMP inhibitors, bone catabolic antagonists, etc. diabetic mice are labeled as being in the right column and WT mice are labeled as being in the left column inFIG.1(C).

The hedgehog agent may be delivered directly to the site for skeletal regeneration. The hedgehog agent may be provided immediately before, during or after the implant is introduced, shortly following a fracture or bone trauma, etc., and in some embodiments is delivered within 1, 2, 3, 4, 5, 6, 7 days following introduction. The hedgehog agent may be transiently provided over a short, defined period of time, for example as a localized implant releasing an active agent for a short period of time, e.g. not more than about 7 days, not more than 6 days, not more than 5 days, not more than 4 days, not more than 3 days, and the like.

In some embodiments of the invention an individual selected for treatment has metabolic syndrome. In some embodiments an individual selected for treatment has type I diabetes. In some embodiments an individual selected for treatment has type II diabetes. In embodiments an individual is tested for indicia of diabetes or metabolic syndrome prior to treatment, including without limitation testing insulin levels, blood sugar levels, hemoglobin A1c levels and the like, as known in the art.

