Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-12-14
2002-09-24
Low, Christopher S. F. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S04400A, C530S350000, C435S006120, C435S091200, C435S069100, C435S325000, C435S374000, C435S378000, C424S422000, C424S049000, C424S534000, C424S078310, C424S078310, C424S078310, C424S078310
Reexamination Certificate
active
06455495
ABSTRACT:
BACKGROUND OF THE INVENTION
Whole bone, which comprises bone tissue, soft tissue, blood vessels and nerves, functions as both a skeletal unit and a physiological unit. Bone tissue is formed of bone cells in an extracellular collagenous matrix. The matrix contains collagen and an inorganic mineral phase called apatite or hydroxyapatite consisting primarily of calcium phosphate crystals. Hydroxyapatite helps provide the necessary stiffness of bone.
Hydroxyapatite has been found to bind tightly to proteins, with calcium sites on the hydroxyapatite appearing to bind acidic groups, e.g., carboxy and phosphate groups, and phosphate sites appearing to bind basic groups. A separation technique known as hydroxyapatite chromatography is based on the interaction of hydroxyapatite with proteins. A variety of hydroxyapatite binding proteins are known to be present in mammalian mineral tissue. These proteins have two known types of binding sites, i.e., one site is believed to be composed of phosphorylated amino acids and the other site is believed to be composed of &ggr;-carboxy glutamic acid. A third type of hydroxyapatite binding site has been hypothesized to consist of consecutive sequences of acidic amino acids. Aspartic acid hexamer (Asp
6
) and glutamic acid hexamer (Glu
6
) were shown to significantly adsorb onto hydroxyapatite, with the adsorption of polyglutamic acid decreasing with a reduction in the number of Glu sequences (R. Fujisawa, et al.,
Biochimica et Biophysica Acta
., 1292:53-60 (1996)). Glu
6
and conjugates of Glu
6
with bovine serum albumin and hemoglobin have been shown to affect the mineralization of hydroxyapatite in the presence of calcium and phosphate (Id.).
Compounds which target and securely bind to bone are of interest for the diagnosis and treatment of diseases of bone. Bone targeting compounds are well known, and include tetracyclines and fluoride. One group of bone targeting compounds, bisphosphonates, have been conjugated with radionuclides and used in skeletal scintillography for the imaging of bone metastases. Bisphosphonates used for bone scanning include hydroxyethylidene diphosphonate, methylene diphosphonate (MDP), hydroxymethylene diphosphonate, and dicarboxypropane diphosphonate, with MDP being the most widely used agent. Additionally, conjugates of bisphosphonates with the antitumor agent methotrexate were found to behave like bone targeting agents with methotrexate being available to treat osteosarcoma (F. Hosain,
J. Nucl. Med
., 37:105-107 (1996)).
In addition to targeting bone, bisphosphonates function by inhibiting bone resorption through their effect on both osteoblasts and osteoclasts, the bone cells responsible for bone growth and bone resorption, respectively. While the mechanism of action of bisphosphonates on bone is not completely understood, it is clear that it is not entirely a matter of adsorption. Moreover. when given in high doses such as those used to prevent ectopic calcification or ossification, bisphosphonates inhibit the mineralization of normal calcified tissues, which results in rickets and osteomalacia. Renal failure can occur due to formation of insoluble aggregates in blood following rapid intravenous injection of large doses of bisphosphonates. Additionally, a strong bond is formed between bisphosphonates and hydroxyapatite, and it has been suggested that, as bisphosphonates slow the resorption of the bone on which they are deposited, their skeletal retention may approach an entire lifetime. Such a long retention time is disadvantageous in applications where a more controlled and transient bond is required.
It would be a significant advantage to have available methodology and compositions for delivering therapeutic agents and imaging agents to bone tissue, wherein the affinity and retention time on the bone can be easily controlled and varied. The present invention meets these and other needs.
SUMMARY OF THE INVENTION
One aspect of this invention provides methods and compositions for delivery of therapeutic agents to bone tissue employing conjugates of negatively charged peptide oligomers.
Another aspect of this invention provides methods and compositions for recruiting endogenous bone affecting agents present in a subject's body fluid to a subject's bone tissue, employing negatively charged peptide oligomers conjugated with moieties capable of binding the endogenous bone affecting agents.
Another aspect of the invention provides methods and compositions for imaging bone tissue or calcified masses employing conjugates of negatively charged peptide oligomers with imaging agents.
A further aspect of this invention provides methods and compositions useful for separating a substance of interest from a solution, employing an adsorption support comprising hydroxyapatite or other anion exchange materials bound to a conjugate of a negatively charged peptide oligomer with a moiety capable of binding the substance of interest.
Also provided are pharmaceutical compositions for therapeutic agent delivery, endogenous substance recruitment, and bone tissue or calcified mass imaging.
The negatively charged peptide oligomers employed in the invention have an affinity for calcified tissue, particularly the hydroxyapatite component of bone tissue, thereby acting as bone targeting carriers for therapeutic agents with which they are conjugated.
In one aspect of the invention the therapeutic agents are delivered to cell surface receptors associated with bone tissue.
The peptide oligomer binds to bone tissue with an affinity that is dependent on the total negative charge of the oligomer, and on the ratio of total negative charge to number of amino acid residues in the oligomer. Accordingly, in one aspect, this invention provides conjugates with an affinity for the hydroxyapatite component of bone tissue or calcified mass, which affinity can be controlled by adjusting the total negative charge and size of the peptide oligomer.
In another aspect of the invention, the rate of degradation of the negatively charged peptide oligomer can be controlled by introducing at least one D-amino acid residue, so that resistance to enzyme cleavage of peptide bonds involving D-amino acids is provided.
Therapeutic agents and endogenous bone affecting agents embraced in the present invention include agents that affect the metabolism or health of bone tissue, thereby providing methods and compositions useful in the treatment or prevention of bone diseases such as, for example, osteosarcoma and osteoporosis.
DETAILED DESCRIPTION OF THE INVENTION
Abbreviations and Definitions
The one- and three-letter abbreviations used herein for the various common amino acids are as recommended in
Pure Appl. Chem
. 31, 639-645 (1972) and 40, 277-290 (1974) and comply with 37 CFR §1.822 (55 FR 18245, May 1, 1990). The abbreviations represent L-amino acids unless otherwise designated as D- or D,L-. Certain amino acids, both natural and non-natural, are achiral, e.g. glycine. All peptide sequences are presented with the N-terminal amino acid on the left and the C-terminal amino acid on the right.
The term “peptide oligomer” refers to a segment of at least about 3 amino acids, up to about 20 to about 50 amino acids, and does not include any natural proteins.
The term “negatively charged peptide oligomer” refers to a peptide oligomer of amino acid residues, wherein one or more amino acids, typically at least three amino acids, are negatively charged amino acids.
The term “negatively charged amino acid” refers to a natural or non-natural amino acid, regardless of chirality, containing, in addition to the C-terminal carboxyl group, at least one additional negatively charged group such as carboxyl, phosphate, phosphonate, sulfonate, or the like.
The term “conjugate” refers to and embraces a negatively charged peptide oligomer linked via a covalent bond to a therapeutic agent, moiety, or imaging agent, such as those described below, wherein such linkage is formed directly or indirectly via a linking agent.
Preferred Embodiments
The invention provides conjugates of negative
Chu Barbara Chen Fei
Orgel Leslie
Foley & Lardner
Low Christopher S. F.
Reiter Stephen E.
Robinson Hope A.
The Salk Institute for Biological Studies
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