Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-01-19
2001-02-20
Russel, Jeffrey E. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S013800, C514S014800, C530S324000, C530S326000, C530S327000, C530S345000
Reexamination Certificate
active
06191110
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method of enhancing wound healing by stimulating fibroblast and keratinocyte growth in vivo, to a method of stimulating the accelerated growth of dermal tissue in a tissue culture containing same, utilizing amphipathic peptides, and to novel synthetic amphipathic peptides for carrying out such methodology.
2. Description of the Related Art
The use of topical antibacterial agents in conjunction with early wound excision and grafting has dramatically reduced the incidence of burn wound sepsis following massive thermal injury.
However, a major disadvantage associated with the use of the most effective topical antimicrobial agents such as Sulfamylon® and Silvadene® in burn or other wound treatment, is that such antimicrobial agents are cytotoxic to fibroblasts and keratinocytes. In consequence, these antimicrobial agents, while efficacious to reduce wound sepsis, in fact oppose dermal/tissue growth and regeneration to an unsatisfactory extent.
Accordingly, it would be a significant advance in the art, and is accordingly an object of the present invention, to provide a therapeutic agent for the treatment of wounds which promotes dermal/tissue growth and regeneration, and which may be used in conjunction with the aforementioned topical antimicrobial formulations.
Other objects and advantages of the present invention will be more fully apparent from the ensuing disclosure and appended claims.
SUMMARY OF THE INVENTION
The present invention relates to a method of treating wounds of mammalian subjects in need of such treatment, to promote wound healing thereof, comprising administering to the subject, e.g., by topically administering to the wound locus, a composition comprising a fibroblast and keratinocyte proliferatingly effective amounr of an amphipathic peptide.
Preferably, the amount of the aephipathic peptide is a lso antimicrobially effective in amount, since the amphipathic peptides are surprisingly and unexpectedly microbicidal in character at concentrations which are in the general range of those which wound-healing promotion effects of such peptides are exhibited.
As used herein, the term “fibroblast and keratinocyte proliferatingly effective amount of an amphipathic peptide” means an amount of an amphipathic peptide which in application to the wound locus functions to promote the growth (as measured by increase in cell count) of fibroblasts and keratinocytes.
The growth of fibroblasts and keratinocytes may readily be determined by cell counts of these cells using a conventional cell counter means, e.g., a flow cytometer or a Coulter counter (Coulter Electronics, Inc., Hialeah, Fla.) in a culture of such cells treated with one or more amphipathic peptides, as measured against a corresponding culture sample of the same type cells, which is not treated with the amphipathic peptide(s).
As used herein, the term “antimicrobially effective” means that the amphipathic peptide is microbicidal at the wound locus to bacteria selected from the group consisting of
Staphylococcus aureus, Pseudomonas aeruginosa
,
Enterococcus species,
and
Xanthomonas maltophilia
, and mixtures thereof. The microbicidal character of the amphipathic peptide(s) to these bacterial species can be readily determined by the analytical method set out hereinafter in Example 1 hereof.
As used herein, the term “amphipathic” in application to a peptide or class of peptides means peptide(s) which contain hydrophilic and hydrophobic amino acid moieties (side chains) which are oriented in relation to one another so that the peptide(s) have discrete hydrophilic and hydrophobic faces or regions defined by a multiplicity of the respective hydrophilic and hydrophobic side chains. For example, when the peptide is in an amphipathic alpha-helix conformation, the hydrophobic amino acid side chains are oriented on one face of the alpha helix while the hydrophilic amino acid side chains are oriented on the other face of the alpha helix. When the peptide is amphipathic and exists (in solution) in a beta-pleated sheet conformation, the peptide likewise exhibits hydrophobic and hydrophilic faces deriving from the alignment of the oriented amino acid side chains of the molecule.
As used herein, the term “defensin-class peptide” means either a natural defensin peptide which is provided in isolated form as an active ingredient of the composition employed for wound healing treatment in accordance with the present invention, or else a synthetic peptide which is homologous to the natural defensin peptide, containing between 17 and 39 amino acids along its length, and forming amphipathic beta-pleated sheets in solution.
A wide variety of amphipathic peptides may be effectively utilized in the broad practice of the present invention, including, but not limited to, natural and synthetic melittin-class, cecropin-class, magainin-class, and defensin-class peptides.
The beta pleated sheet conformation of peptides potentially usefully employed in the broad practice of the present invention may be readily determined by the circular dichroism technique described in Proteins, Creighton, Thomas E., W.H. Freeman & Co., New York (1984), pp. 179-182.
In another aspect, the present invention relates to a method of stimulating the accelerated growth of dermal tissue in a tissue culture containing same, comprising applying to the tissue culture a fibroblast and keratinocyte proliferatingly effective amount of an amphipathic peptide. Such methodology may for example be employed to produce skin for skin grafting purposes, utilizing a dermal tissue culture containing dermal tissue material of a skin graft recipient.
Other aspects and features of the present invention will be more fully apparent from the ensuing disclosure and appended claims.
DETAILED DESCRIPTION OF THE INVENTION, AND PREFERRED EMBODIMENTS THEREOF
The disclosures of prior copending U.S. patent application No. filed Apr. 8, 1994 in the names of Jesse M. Jaynes and Gordon R. Julian for “METHOD OF COMBATING MAMMALIAN NEOPLASIA, AND LYTIC PEPTIDES THEREFOR,” now U.S. Pat. No. 5,773,413 U.S. patent application Ser. No. 08/039,620 filed Jun. 4, 1993 in the names of Jesse M. Jaynes and Gordon R. Julian, now U.S. Pat. No. 5,744,445 U.S. patent application Ser. No. 08/148,889 filed Nov. 8, 1993 in the name of Gordon R. Julian, now U.S. Pat. No. 5,717,069 and U.S. patent application Ser. No. 08/148,491 filed Nov. 8, 1993 in the name of Gordon R. Julian, now U.S. Pat. No. 5,968,904 are all hereby incorporated herein by reference in their entirety.
The present invention is based on the surprising and unexpected discovery that amphipathic peptides may be advantageously employed to stimulate the proliferative growth of fibroblasts and epithelial cells such as keratinocytes, to thereby effect enhanced wound healing in mammalian subjects, and the further surprising and fortuitous finding that the amphipathic peptides have such dermal call growth-promoting properties at concentrations which concomitantly have antimicrobial efficacy, against microbial species including those which cause or otherwise mediate sepsis and wound infection.
Defensins are a particularly preferred class of natural and synthetic peptides that are usefully employed in the practice of the invention and that have been discovered to possess a broad bactericidal spectrum as well as being mitogenic for fibroblasts and epithelial cells. These defensin-class peptides are arranged over at least a portion of their length (of amino acid sequence) so that the sucessive amino acid side chains are alternatingly hydrophobic (on one face of the molecule) and hydrophilic (on the other face of the molecule).
Naturally occurring amphipathic peptides play an important if not critical role as immunological agents in insects and have some, albeit secondary, defense functions in a range of other animals. The function of these peptides is to destroy prokaryotic and other non-host cells by disrupting the cell membrane and promoting cell lysis. Common features of these
Jaynes Jesse M.
Julian Gordon R.
Demegen, Inc.
Rothwell Figg Ernst & Manbeck
Russel Jeffrey E.
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