Peptides for altering osteoblast adhesion

Details Peptides for altering osteoblast adhesion Peptides for altering osteoblast adhesion

1998-07-14

2001-07-17

Jones, Dwayne C. (Department: 1614)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S015800, C514S016700, C514S017400, C514S018700, C530S324000, C530S325000, C530S326000, C530S327000, C530S328000, C530S329000, C530S330000, C623S011110, C623S016110

Reexamination Certificate

active

06262017

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to peptides containing a cell adhesion-related amino acid sequence, and to devices and biomaterials for implantation coated with or incorporating peptides containing that sequence.
BACKGROUND OF THE INVENTION
Improved understanding of cellular and molecular events which occur at the interface between tissues and implants is beginning to allow new approaches to biomaterial design. The challenge is to produce biomaterials that are engineered to elicit specific, clinically-desirable responses from living cells and tissues in a patient's body. For example, it is clinically desirable for osteoblasts to rapidly deposit mineralized matrix on the surface of (or in close apposition to) newly implanted prostheses. The swift deposition of bone stabilizes the prosthesis and minimizes motion-induced damage to surgically traumatized tissue at the implantation site.
Anchorage-dependent cells (such as osteoblasts) must first adhere to a surface in order to perform subsequent cellular functions (e.g., proliferation, deposition of bone tissue, etc.). Since cell adhesion is the key to subsequent events, methods for promoting cell adhesion are of considerable interest. The effects on cell adhesion of peptides immobilized on the surfaces of substrates have been reported. Substrates have included polymers [Massia and Hubbell
Journal of Biomedical Materials
25, 223-242 (1991)] and dental/orthopedic implant materials such as Cobalt-Chromium-Molybdenum alloy [Mikos et al.
Biomaterials for Cell and Drug Delivery
331, 269-274 (1994)]. Adhesion-related peptides that have been attached to substrates have been mainly integrin-binding peptides, such as those which contain the Arginine-Glycine-Aspartic Acid (RGD) sequence.
The present invention relates to novel, heparin-binding, osteoblast-adhesive amino acid sequences of the formula:
aa
1
-aa
2
-aa
3
-aa
4
wherein:
aa
1
represents the residue of an amino acid selected from the group consisting of H-Lys, H-Arg, H-Orn, and 6-aminocaproic acid (Acp);
aa
2
, and aa
4
independently represent the residue of an amino acid selected from the group consisting of lysine (Lys), arginine (Arg), and ornithine (Orn); and
aa
3
represents the residue of an amino acid selected from the group consisting of alanine (Ala), glycine (Gly), valine (Val), leucine (Leu), isoleucine (Ile), serine (Ser), threonine (Thr), cysteine (Cys), methionine (Met), asparagine (Asn), norleucine (Nle), norvaline (Nva), and 2-aminobutyric acid (Abu). This peptide sequence has not been previously shown to exhibit any biological activity.
SUMMARY OF THE INVENTION
In one aspect, the invention relates to compounds of formulae
Aa
1
-aa
2
-aa
3
-aa
4
-(Gly)
n
—OH
and
[Aa
1
-aa
2
-aa
3
-aa
4
]
4
-MAP
wherein:
Aa
1
represents the residue of an amino acid selected from the group consisting of H-Lys, H-Arg, H-Orn, and 6-aminocaproic acid (Acp);
aa
2
, and aa
4
independently represent the residue of an amino acid selected from the group consisting of lysine (Lys), arginine (Arg), and ornithine (Orn);
aa
3
represents the residue of an amino acid selected from the group consisting of alanine (Ala), glycine (Gly), valine (Val), leucine (Leu), isoleucine (Ile), serine (Ser), threonine (Thr) cysteine (Cys), methionine (Met), asparagine (Asn), norleucine (Nle), norvaline (Nva), and 2-aminobutyric acid (Abu); and n is zero or an integer from 1 to 6. The invention also includes pharmaceutically acceptable salts of the foregoing compounds. Under certain circumstances, discussed below, tetrapeptides of the following formulae are preferred: H-Lys-Arg-Met-Arg-OH (n=0 SEQ ID NO:12); H-Lys-Arg-Ala-Arg-OH (n=0 SEQ ID NO:19); H-Arg-Arg-Ser-Arg-OH (n=0 SEQ ID NO:26); H-Orn-Arg-Ser-Arg-OH (n=0 SEQ ID NO:33); H-Lys-Lys-Ser-Lys-OH (n=0 SEQ ID NO:40); H-Acp-Arg-Ser-Arg-OH (n=0 SEQ ID NO:47). The peptide sequence which is most preferred is H-Lys-Arg-Ser-Arg-OH (i.e. n=0 SEQ ID NO:1). The single-letter code for Lys-Arg-Ser-Arg is KRSR.
In another aspect, the invention relates to a device for implantation in an animal. The device comprises a substrate covalently attached to a peptide that includes the cell adhesion-related sequence and that has a molecular weight less than 4 kDa. The substrate may be a ceramic, a metal, a polymer, a polymer-coated or glass-coated metal or a composite. A preferred peptide has the formula H-Lys-Arg-Met-Arg-(Gly)
3
—OH; H-Lys-Arg-Ala-Arg-(Gly)
3
—OH; H-Arg-Arg-Ser-Arg-(Gly)
3
—OH; H-Orn-Arg-Ser-Arg-(Gly)
3
—OH; H-Lys-Lys-Ser-Lys-(Gly)
3
—OH; or H-Acp-Arg-Ser-Arg-(Gly)
3
—OH, with H-Lys-Arg-Ser-Arg-(Gly)
3
—OH and [KRSR]
4
-MAP being most preferred. The peptide may be attached to the substrate through a spacer chosen from the group consisting of poly(glycine), poly(alanine), poly(ethyleneimine), poly(lysine), hydroxyethylcellulose, poly(ethylene glycol), &agr;,&ohgr;-alkylenediamines and &ohgr;-aminoalkanoic acids. A bone prosthetic device is a particularly preferred embodiment of the device aspect of the invention.
In another aspect, the invention relates to a method for enhancing the stabilization of an implant. According to the method, the implant is provided with a coating of a peptide of molecular weight less than 4 kDa incorporating the cell adhesion-related sequence. Preferably the coating of peptide is covalently attached to the implant. The preferred peptides are as above.
In a closely related aspect, the invention relates to a method for promoting the adhesion of osteoblasts to a prosthetic device. The method comprises providing a peptide of molecular weight less than 4 kDa incorporating the cell adhesion-related sequence at the surface of the prosthetic device. Preferred peptides are as above. The most preferred peptide is H-Lys-Arg-Ser-Arg-(Gly)
n
—OH.
In yet another aspect, the invention relates to a method for disrupting cell adhesion. The method comprises bringing a cell into contact with a compound containing the cell adhesion-related peptide sequence at a concentration sufficient to inhibit cell adhesion.
In a closely related aspect, the invention relates to a method for treating a disease associated with cell adhesion. The method comprises administering to a mammal suffering from such a disease an amount of a compound containing the cell adhesion-related peptide sequence sufficient to inhibit cell adhesion.
In yet another aspect, the invention relates to a method of constructing a bone replacement or bone-reconstructive material. The method comprises preparing a biodegradable or inert polymer matrix which incorporates the cell adhesion-related peptide sequence, bringing osteoblasts into contact with the polymer matrix and adding growth factors.


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Cardin et al. “Molecular Modeling of Protein-Glycosaminoglycan Interactions”Arteriosclerosis 9, 21-32 (1989).
Puleo et al. “Mechanisms of fibronectin-mediated attachment of osteoblasts

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