The p97 protein and transferrin were also found to be expressed in brain capillary endothelial cells in both normal controls and pathological brains. Most of the p97 molecule detected was intracellular and its expression was coincidental with the transferrin receptor. Electron microscopic examination also indicates that p97 crosses the blood brain barrier. p97 has also been shown to bind to a soluble form of transferrin receptor. The results of affinity chromatography experiments suggest that there is a receptor which co-recognizes p97 and the transferrin receptor.

Taken together, these findings suggest that p97 may be used to modulate iron uptake in cells. This could be accomplished by regulating ing the concentration of p97, inhibiting p97 binding to iron or to the transferrin receptor, or inhibiting binding to the receptor which co-recognizes p97 and the transferrin receptor. Accordingly, p97, and stimulants, agonists or antagonists of p97 may be useful in the treatment of conditions where there is a disturbance in iron metabolism. For example, such substances may be useful in the treatment of conditions such as haemochromatosis, neurodegenerative diseases, ischemic tissue damage, including ischemic stroke or trauma, heart disease and tumours, in particular skin cancer (melanoma).
The discovery of a role for p97 in iron uptake and iron transport and in particular the finding that p97 can cross the blood brain barrier, suggests that p97 can be used to transport substances such as therapeutic agents across the blood brain, blood eye or blood placenta barriers.

Importantly, the Inventors have shown that Alzheimer’s patients have elevated levels of p97 in their plasma, serum and cerebrospinal fluid and that p97 levels increase with duration of the disease. The levels of p97 in patients’ samples may potentially be used to diagnose and to monitor the progression of the disease and the efficacy of therapeutic treatments for Alzheimer’s disease.

The Inventors also have found that reactive brain microglial cells associated with senile plaques in Alzheimer’s disease express p97 and transferrin receptor. Therefore, p97 and transferrin receptor can be used in the diagnosis of Alzheimer’s disease. The finding that microglial cells which deposit the amyloid protein have a high level of proteins which operate in procurement of iron also suggests methods of treatment of Alzheimer’s disease based on depletion of iron from these cells using substances such as p97, transferrin, and iron chelators, for example, lactoferrin, ferritin and ovotransferrin.

The discoveries achieved by Dr. Jefferies and his research team have led to a large body of intellectual property related broadly to p97. What follows is a summation of the “inventions surrounding p97” and its use in various neurological diseases, including Alzheimer’s disease. These will include the use of p97 as a biomarker for diagnosis, for transport of therapeutic molecules across various biological barriers (including the blood-brain barrier), and for control of iron metabolism in a variety of neurological disorders.

The invention further relates to a method for identifying a stimulant, agonist or antagonist of p97-mediated iron uptake comprising: incubating a cell expressing p97 on its surface and a substance suspected of being a stimulant, agonist or antagonist of p97 in the presence of iron and in the absence of transferrin, measuring the amount of iron uptake into the cell and identifying the stimulant, agonist or antagonist of p97-mediated iron uptake by comparing the amount of iron uptake in the cell with the amount of iron uptake in a cell from a control incubation in the absence of the substance.

In an embodiment, the cell is incubated in the presence of labeled iron and the amount of iron uptake in the cell is determined by measuring the amount of labeled iron in the cell. The label may be, for example, radioactive or fluorescent.
The invention also relates to a composition for delivering an agent across the blood brain barrier comprising p97 or a substance which is capable of specifically binding to p97, in association with the agent and a pharmaceutically acceptable carrier or diluent. The p97 or substance, preferably antibody to p97, may be conjugated to the agent or a p97 fusion protein may be used in the composition. The agent may be a substance having therapeutic activity such as a growth factor or lymphokine. The invention also relates to a method of delivering an agent across the blood brain barrier comprising administering the agent in association with p97 or antibody to p97. The composition of the invention may also be used for delivering an agent across the blood eye or blood placenta barrier.

Within one aspect of the present invention, a composition for the preservation of organs intended for transplantation is provided comprising p97 or a derivative thereof in a pharmaceutically acceptable organ preservation solution. The invention also contemplates a method for preserving an organ intended for transplantation using the composition.

The present invention also provides methods for diagnosing and monitoring Alzheimer’s disease, as well as compositions and methods suitable for treating Alzheimer’s disease. Within one aspect of the present invention, methods are provided for monitoring Alzheimer’s disease, comprising detecting the presence of soluble p97 in a patient. Within various embodiments, the p97 may be detected in various bodily fluids, including for example, urine, blood, serum and cerebral spinal fluid. Various methods may be utilized to detect p97, including, for example, radioimmunoassay, competitive assays, and enzyme linked immunosorbent assays (“ELISA”) such as the antigen-capture “sandwich assay”. Within other aspects of the present invention, methods are provided for monitoring Alzheimer’s disease comprising detecting the presence of transferrin receptors, and/or detecting the presence of p97, on microglial cells associated with amyloid plaques in a patient.

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