Controlled release of non heparin-binding growth factors...

Details Controlled release of non heparin-binding growth factors... Controlled release of non heparin-binding growth factors...

2001-05-03

2004-04-20

Eyler, Yvonne (Department: 1646)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Matrices

C424S078100, C424S085100, C424S279100, C530S300000, C514S002600, C514S056000

Reexamination Certificate

active

06723344

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the field of three-dimensional matrices that contain pharmacologically active molecules, particularly growth factors. The invention also relates to the use of growth factors or proteins in a matrix designed to promote cell and tissue growth. The invention further relates to the use of growth factors with low heparin-binding affinity. In addition, the invention relates to the field of articles of manufacture useful as implantable devices and wound dressings as the matrix of the invention is designed to be used in conjunction with such devices to provide protracted and controlled release of growth factor, thus promoting wound healing in the patient.
BACKGROUND
Many growth factors are thought of as “heparin-binding” growth factors. Families with one or more members that bind heparin include fibroblast growth factors and bone morphogenetic proteins (BMPs) (1, 2). Additional growth factors that bind heparin include transforming growth factor &bgr;1 (TGF-&bgr;1), interleukin-8, neurotrophin-6, vascular endothelial cell growth factor, heparin-binding epidermal growth factor, hepatocyte growth factor, connective tissue growth factor, midkine, and heparin-binding growth associate molecule (3-11). These factors have shown the potential to enhance healing in many different types of tissue including vasculature, skin, nerve, and liver.
Controlled delivery devices based on heparin-affinity of these growth factors have been designed previously (12-14). These drug delivery devices have previously been used to deliver “heparin-binding” growth factors. Such “heparin-binding” growth factors are typically considered to be those which bind to heparin with a relatively high affinity, often characterized by elution from heparin-affinity columns at NaCl concentrations well above physiological levels (>140 mM). In such delivery systems, the heparin-binding affinity of the growth factor is usually used to sequester the growth factors to immobilized heparin of some form. For example, Edelman et al. have used heparin-conjugated Sepharose beads to bind basic fibroblast growth factor (bFGF) and then encapsulated the beads with alginate (12, 19). These beads serve as reservoirs that release bFGF slowly based on the binding and dissociation constants of bFGF and heparin.
The delivery of “non-heparin-binding growth factors” has previously required release methods for delivery typically based on diffusion-controlled release of the factors from porous materials (15-18). There remains a need in the medical arts for a device that is capable of providing the release of low heparin-binding growth factors at a controlled and predictable rate in order-to provide effective release of the factor over a clinically useful period during the wound healing process.
SUMMARY OF THE INVENTION
In a general and overall sense, the present invention relates to the use of how non-heparin-binding growth factors in delivery techniques employing growth factors with heparin-affinity by utilizing low heparin affinity sequences present in many proteins. The particular growth factors employed as part of the invention have been found by the present inventors to possess a basic sequence at a site in the protein that is freely accessible in the proteins native conformation. This basic region may possess only relatively low heparin-affinity.
As used in the description of the present invention, “low-heparin-binding affinity” of a growth factor or peptide fragment thereof is defined as any protein, peptide, or derivative or combination thereof, that is capable of demonstrating the biological activity of a growth factor, and that has a relatively binding low affinity for binding heparin, and will elute from a heparin-affinity column at sub-physiological NaCl concentrations. Physiological levels of NaCl may be defined as about 140 mM NaCl. Herein the term “sub-physiological” levels of NaCl, therefore, may be further defined as from between about 25 mM to about 140 mM NaCl. Although low heparin-binding affinity growth factors elute from heparin-affinity columns at sub physiological NcCl concentrations, their low affinity for heparin can still be used to sequester the protein or peptide to a matrix that contains heparin or a heparin-binding site.
By way of example, and in no way intending to be limited to any particular mechanism of action, the invention may be described as employing a matrix having growth factor proteins with a relatively high-ratio of heparin-binding sites. A ratio of at least 1:1 heparin to growth factor must be used, but the greater the excess of heparin sites the slower the release. In this fashion, primarily “non-heparin-binding” growth factors or peptide fragments thereof with relatively low heparin-binding affinity can be bound to a heparin-decorated matrix. These matrices can then serve as reservoirs containing the growth factor or factors to be delivered. The dissociation kinetics of low affinity heparin-binding proteins are relatively fast, but the high number of binding sites allows rebinding of the growth factor before it can diffuse out of the matrix. Release can occur by diffusion of the growth factor out of the matrix prior to rebinding, or it can occur if the growth factor encounters a cell surface receptor before rebinding to a heparin site. In this fashion, release of the growth factor or bioactive fragment thereof can be sustained, and continue to foster improved healing.
In a general and overall sense, the present invention describes in at least one aspect a specially designed matrix that provides for the release of growth factors or bioactive fragments thereof. The growth factor is defined as having low binding affinity for heparin. The matrix, more particularly, may be defined as comprising a substrate capable of providing attachment of heparin, a heparin-like polysaccharide, or a heparin-like polymer, and a growth factor or peptide fragment thereof having a basic domain that binds heparin with low affinity.
The characteristic of the growth factor or peptide fragment thereof as binding to heparin with low affinity may be further described as a peptide/protein that will elute from a heparin affinity column at an NaCl concentration of about 25 mM to about 140 mM.
The “low heparin-binding affinity” growth factor or peptide fragment thereof may be further defined as comprising a length of about 8 to 30 amino acid residues. This sequence of amino acid residues, in some embodiments, may be defined as comprising at least 2 basic amino acid residues, a ratio of basic to acidic amino acid residues of at least 2, and a ratio of hydrophobic amino acid residues to basic amino acid residue of at least 0.67. The growth factor or fragment thereof elutes from a heparin affinity column at less than 140 mM or at about 25 to aboutl 40 mM NaCl.
For purposes of this application, basic amino acids may be defined as K (lysine) or R (arginine). The acidic amino acid residues may be further defined as D (aspartic acid) or E (glutamic acid). The hydrophobic amino acid residues may be defined as A (alanine), V (valine), F (phenylalanine), P (proline), M (methionine), I (isoleucine), or L (leucine). For purposes of this application, C (cysteine) that are involved in a disulfide bridge are also considered hydrophobic.
By way of example, the low heparin-binding affinity growth factor or a peptide fragment thereof as defined in the invention comprises neurturin, persephin, IGF-1A, IGF-1&bgr;, EGF, NGF&bgr;, NT-3, BDNF, NT-4, TGF-&bgr;2, TGF-&bgr;3, TGF-&bgr;4, or a peptide fragment of any one of these. Other growth factors may be found which contain similar basic domains that are not enumerated here. The matrix itself may also comprise any of a variety of materials, such as fibrin, collagen, hyaluronic acid, or a synthetic polymer hydrogel. By way of example, the synthetic polymer hydrogel may be a poly (ethylene glycol) hydrogel or a derivative thereof. Other synthetic polymer hydrogels may be used apart from those enumerated here.
The peptides of the invention that bind heparin

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