Drug – bio-affecting and body treating compositions – Effervescent or pressurized fluid containing
Reexamination Certificate
1999-09-01
2001-06-12
Russel, Jeffrey E. (Department: 1653)
Drug, bio-affecting and body treating compositions
Effervescent or pressurized fluid containing
C424S045000, C424S400000, C514S002600, C514S012200, C514S013800, C514S014800, C514S015800, C514S016700, C514S017400
Reexamination Certificate
active
06245320
ABSTRACT:
BACKGROUND OF THE INVENTION
Hypersecretion of mucin in the airways is associated with a variety of diseases, including asthma, chronic bronchitis, cystic fibrosis, and bronchiectasis. Effective measures for inhibiting mucin secretion in the airways would be useful to mitigate the deleterious effects associated with mucin hypersecretion. Effective inhibition of mucin secretion would also be useful in enhancing the delivery of therapeutic agents to the airways and via the airways.
SUMMARY OF THE INVENTION
It has been discovered, according to this invention, that polycationic peptides inhibit mucin secretion from airway goblet cells. It has also been discovered that the inhibition of mucin secretion by polycationic peptides is accompanied by minimal cytotoxicity to cells of airways, thereby by indicating the suitability of polycationic peptides for use in animals, particularly humans, for the inhibition of mucin secretion. It has also been discovered that polycationic peptides can prevent SO
2
induced goblet cell metaplasia.
It is contemplated as part of this invention that polycationic molecules, particularly polycationic peptides or peptide mimetics, may be employed to inhibit mucin secretion for a variety of therapeutic purposes. For example, the polycationic molecules may be administered to alleviate mucin hypersecretion, particularly in disease conditions associated with mucin hypersecretion. For example, the compositions of the invention may be administered to treat asthma, chronic bronchitis, cystic fibrosis, bronchiectasis, and chronic obstructive pulmonary disease. As another example, the polycationic molecules may be administered to reduce mucin in the airways in order to facilitate the bioavailability of therapeutic agents targeted to the airways, such as, for example, bronchodialators. As another example, the polycationic molecules may be administered to reduce mucin in the airways, thereby minimizing airway impedance and facilitating therapeutic agent delivery to the alveoli or through the alveoli to the blood stream.
Treat, treated or treatment, as used herein, means the administration of polycationic molecules to the airways of an animal to prevent, mitigate, alleviate or cure a disease or the symptoms of a disease.
Animal, as used herein, means any vertebrate animal, including humans, farm animals and pets.
Airways or airway, as used herein, refers to any part of the lungs that is capable of mucin production.
Polycationic molecules that may be used in accordance with this invention include, but are not limited to, polycationic peptides and polycationic peptide mimetics. Polycationic peptides are a preferred polycationic molecule because they can be easily degraded to amino acids.
The polycationic peptides typically comprise about 5 to about 60 amino acids, preferably about 5 to about 40 amino acids, and more preferably about 10 to about 25 amino acids. Smaller molecules are preferred because of greater ease of handling. A polycationic peptide typically possesses a sufficient number of positively charged amino acids such that the pKa of the peptide is greater than 9.0, preferably greater than 10.0, and more preferably greater than 11.0. Typically, at least about 20% of the amino acid residues are positively charged, preferably at least about 40%, more preferably at least about 60%, and most preferably at least about 80%. Positively charged amino acids include, for example, lysine, arginine, or ornithine. Positively charged refers to the side chains of the amino acids which have a net positive charge at a pH 7.0. The effectiveness of any particular polycationic molecule in inhibiting mucin, with minimal toxicity, may be established, for example, using techniques and models described herein. At an optimal concentration, a polycationic molecule, polycationic peptide, or polycationic peptide mimetic, according to this invention, preferably inhibits mucin secretion at least about 50%, more preferably at least about 70%, and most preferably at least about 90%. The degree of inhibition of mucin secretion may be determined, for example, using a hamster tracheal surface epithelial (HTSE) cell culture system as described in the Examples herein. The degree of inhibition of mucin secretion may also be determined using other techniques, including in vitro techniques such as are described, for example, in Adler et al., 1990
, J. Clin. Invest
. 85:75-85 and in Adler et al., 1992
, Am. J. Respir. Cell Mol. Biol
. 6:550-556 and including in vivo techniques such as are described, for example, in Temann et al., 1997
, Am. J. Respir. Cell Mol. Biol
. 16:471-478 and Fahy et al., 1993
, Am. Rev. Respir
. Dis. 147:1132-1137. A polycationic molecule, polycationic peptide, or polycationic peptide mimetic according to this invention preferably has minimal cytotoxicity. Cytotoxicity may be determined, for example, using an LDH release assay, a
51
Cr release assay, or a cell exfoliation assay as described in the examples. Cytotoxicity may also be determined, for example, using an HTSE cell culture system as described in the examples. In determining cytotoxicity for polycationic molecules of the invention using the above listed assays, LDH release,
51
Cr release, or cell exfoliation is typically less than about 115% of that observed in control cells, preferably less than about 110%, and more preferably less than about 105%.
Preferred polycationic peptides for use in accordance with this invention are poly-L-lysine, poly-L-arginine, or poly-L-lysine and poly-L-arginine heteropolymers. Polycationic peptides containing other positively charged amino acid residues such as, for example, poly-L-ornithine, may also be employed. The amino acids of the polycationic peptides may be naturally occurring proteogenic amino acids as well as non-naturally occurring amino acids such as amino acid analogs. One of skill in the art would know that this definition includes, unless otherwise indicated, naturally occurring proteogenic (D) or (L) amino acids, chemically modified amino acids, including amino acid analogs such as penicillamine (3-mercapto-D-valine), naturally occurring non-proteogenic amino acids such as norleucine and chemically synthesized compounds that have properties known in the art to be characteristic of an amino acid. As used herein, the term “proteogenic” indicates that the amino acid can be incorporated into a protein in a cell through well-known metabolic pathways. The choice of including an (L)- or a (D)-amino acid into a peptide of the present invention depends, in part, on the desired characteristics of the peptide. For example, the incorporation of one or more (D)-amino acids can confer increasing stability on the peptide in vitro or in vivo. As used herein, the term “amino acid equivalent” refers to compounds which depart from the structure of the naturally occurring amino acids, but which have substantially the structure of an amino acid, such that they can be substituted within a peptide which retains biological activity. Thus, for example, amino acid equivalents can include amino acids having side chain modifications or substitutions, and also include related organic acids, amides or the like. The term “amino acid” is intended to include amino acid equivalents. The term “residues” refers both to amino acids and amino acid equivalents.
As used herein, the term “peptide” is used in its broadest sense to refer to compounds containing amino acid equivalents or other non-amino groups, while still retaining the desired functional activity of inhibiting mucin secretion. Peptide equivalents can differ from conventional peptides by, for example, the replacement of one or more amino acids with related organic acids (such as PABA) or the substitution or modification of side chains or functional groups. It is to be understood that limited modifications can be made to a peptide without destroying its biological function, such as for example, the addition of chemical moieties such as amino or acetyl groups.
Polycationic peptides that may be used in accordance with this invention include natur
Millen White Zelano & Branigan
Russel Jeffrey E.
University of Maryland
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