Method for the treatment of pulmonary disease and method of producing proteins of use therein
Inventors
Moss, Joel • Stevens, Linda • Levine, Rodney L.
Assignees
US Department of Health and Human Services
Publication Number
US-8828380-B2
Publication Date
2014-09-09
Expiration Date
2030-09-08
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Abstract
Disclosed herein are methods of treating a subject with pulmonary disease including administering to the subject a therapeutically effective amount of a polypeptide including at least one arginine residue susceptible to ADP-ribosylation and nicotinamide adenine dinucleotide (NAD). In some embodiments, the polypeptide and/or NAD is administered via inhalation. Also disclosed is a pharmaceutical composition including at least one polypeptide (such as HNP-1) and NAD. The disclosure also provides in vitro methods of producing a polypeptide with altered activity, including contacting the polypeptide with NAD and an arginine-specific mono-ADP-ribosyltransferase (for example, ART1) to produce a polypeptide including at least one ADP-ribosylated arginine residue, incubating the ADP-ribosylated polypeptide under conditions sufficient for conversion of at least one ADP-ribosylated arginine residue to ornithine, and isolating the ornithine-containing polypeptide. Methods of treating a subject with pulmonary disease including administering to the subject a therapeutically effective amount of a modified polypeptide (such as HNP-1) including at least one ornithine residue in place of an arginine residue are also disclosed.
Core Innovation
This disclosure relates to methods of treating pulmonary disease by administering therapeutically effective amounts of polypeptides including at least one arginine residue susceptible to ADP-ribosylation and nicotinamide adenine dinucleotide (NAD), optionally with administration via inhalation. It includes methods where an arginine-specific mono-ADP-ribosyltransferase (ART), such as ART1, is also administered to facilitate ADP-ribosylation of the polypeptide. The polypeptides of interest include defensins, specifically alpha defensins such as human neutrophil peptide-1 (HNP-1). The disclosure further relates to pharmaceutical compositions containing such polypeptides and NAD.
A second aspect involves in vitro methods of producing polypeptides with altered activity by contacting polypeptides including arginine residues susceptible to ADP-ribosylation with NAD and ART to form ADP-ribosylated arginine-containing polypeptides, incubating under conditions sufficient to convert ADP-ribosylated arginine residues to ornithine residues, and isolating the ornithine-containing polypeptides. These modified polypeptides, including defensins with ornithine replacing arginine, can be used therapeutically for pulmonary diseases.
The background problem addressed is the cytotoxicity of defensins toward mammalian cells, including human epithelial and endothelial cells, which limits their potential usefulness for treating pulmonary diseases. Existing defensins exhibit antimicrobial activity but can cause undesired cytotoxic side effects. Thus, there is a need for methods to modify these proteins, such as through ADP-ribosylation of arginine residues and/or conversion of modified arginines to ornithine, to modulate their activity, reduce cytotoxicity, and improve therapeutic effectiveness for pulmonary diseases.
Claims Coverage
The claims of the patent include one independent claim focused on a modified human neutrophil peptide-1 polypeptide for treating pulmonary disease, identifying key inventive features related to its sequence and administration.
Modified human neutrophil peptide-1 polypeptide comprising ornithine residues
The claim covers a therapeutic method using a modified HNP-1 polypeptide having the amino acid sequence of SEQ ID NO: 2 where both positions 14 and 24 are replaced with ornithine residues.
Administration via inhalation
The method includes administering the modified HNP-1 polypeptide by inhalation to the subject as a treatment for pulmonary disease.
Treatment of various pulmonary diseases including infections
The pulmonary diseases treated include cystic fibrosis, emphysema, asthma, sarcoidosis, chronic bronchitis, bronchopulmonary dysplasia, pulmonary fibrosis, pneumonia (specifically bacterial pneumonia), adult respiratory distress syndrome, and other pulmonary diseases caused by infections.
Enhanced therapeutic properties of the modified polypeptide
The modified HNP-1 polypeptide exhibits antimicrobial activity with reduced cytotoxicity to mammalian cells compared to unmodified HNP-1, and administration leads to increased cytokine production, notably interleukin-8 (IL-8).
The claims focus on treating pulmonary diseases using a therapeutically effective amount of a modified HNP-1 polypeptide containing ornithine residues at positions 14 and 24, preferably administered by inhalation. This provides a therapy with antimicrobial efficacy, reduced cytotoxicity, and immunomodulatory effects such as increased cytokine production.
Stated Advantages
Modification of defensin polypeptides by ADP-ribosylation and conversion of arginine to ornithine reduces cytotoxicity toward mammalian cells while retaining antimicrobial activity.
Administration by inhalation can deliver therapeutic polypeptides and NAD directly to the pulmonary system, improving treatment for pulmonary diseases.
Modified polypeptides exhibit increased cytokine production, such as IL-8, potentially enhancing immune responses.
Documented Applications
Treatment of pulmonary diseases including cystic fibrosis, emphysema, asthma, sarcoidosis, chronic bronchitis, bronchopulmonary dysplasia, pulmonary fibrosis, pneumonia, and adult respiratory distress syndrome.
Use of polypeptides and pharmaceutical compositions for inhalational administration to subjects with pulmonary disease.
In vitro production of modified polypeptides containing ornithine for use as therapeutic agents in pulmonary disease.
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