U.S. patent application number 14/450512 was filed with the patent office on 2014-11-20 for method for the treatment of pulmonary disease and method of producing proteins of use therein.
This patent application is currently assigned to The United States of America, as represented by the Secretary, Department of Health and Human Serv. The applicant listed for this patent is The United States of America, as represented by the Secretary, Department of Health and Human Serv, The United States of America, as represented by the Secretary, Department of Health and Human Serv. Invention is credited to Rodney L. Levine, Joel Moss, Linda Stevens.
Application Number | 20140341876 14/450512 |
Document ID | / |
Family ID | 43733071 |
Filed Date | 2014-11-20 |
United States Patent
Application |
20140341876 |
Kind Code |
A1 |
Moss; Joel ; et al. |
November 20, 2014 |
METHOD FOR THE TREATMENT OF PULMONARY DISEASE AND METHOD OF
PRODUCING PROTEINS OF USE THEREIN
Abstract
Methods of treating a subject with pulmonary disease by
administering a therapeutically effective amount of a defensin
polypeptide including at least one arginine residue susceptible to
ADP-ribosylation and nicotinamide adenine dinucleotide (NAD), are
described. The polypeptide and/or NAD can be administered via
inhalation. Also disclosed is a pharmaceutical composition
including at least one defensin polypeptide and NAD. In vitro
methods of producing a polypeptide with altered activity, including
contacting the polypeptide with NAD and an arginine-specific
mono-ADP-ribosyltransferase 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, are
also described. Methods of treating a subject with pulmonary
disease by administering a therapeutically effective amount of a
modified defensin polypeptide including at least one ornithine
residue in place of an arginine residue are also disclosed.
Inventors: |
Moss; Joel; (Bethesda,
MD) ; Stevens; Linda; (Gaithersburg, MD) ;
Levine; Rodney L.; (Rockville, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Department of Health and Human Serv |
Bethesda |
MD |
US |
|
|
Assignee: |
The United States of America, as
represented by the Secretary, Department of Health and Human
Serv
|
Family ID: |
43733071 |
Appl. No.: |
14/450512 |
Filed: |
August 4, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13394393 |
Mar 6, 2012 |
8828380 |
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PCT/US2010/048068 |
Sep 8, 2010 |
|
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14450512 |
|
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61241311 |
Sep 10, 2009 |
|
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Current U.S.
Class: |
424/94.5 ;
514/1.5; 514/1.6; 514/1.7; 514/1.8; 530/324 |
Current CPC
Class: |
A61P 11/00 20180101;
C07K 14/47 20130101; A61K 38/1729 20130101; A61K 31/7084 20130101;
A61K 38/45 20130101; A61K 38/17 20130101; A61K 38/16 20130101; A61P
11/06 20180101; C07K 14/4712 20130101; C12Y 204/0203 20130101 |
Class at
Publication: |
424/94.5 ;
514/1.8; 530/324; 514/1.7; 514/1.6; 514/1.5 |
International
Class: |
C07K 14/47 20060101
C07K014/47; A61K 38/45 20060101 A61K038/45; A61K 38/17 20060101
A61K038/17; A61K 31/7084 20060101 A61K031/7084 |
Claims
1. A method of treating a subject with pulmonary disease,
comprising administering to the subject a therapeutically effective
amount of a defensin polypeptide and nicotinamide adenine
dinucleotide (NAD), wherein the defensin polypeptide comprises at
least one arginine residue that is susceptible to ADP-ribosylation,
and wherein the NAD is administered by inhalation.
2. The method of claim 1, wherein the defensin polypeptide
comprises a human neutrophil peptide-1 (HNP-1) polypeptide.
3. The method of claim 2, wherein the HNP-1 polypeptide comprises
the amino acid sequence of SEQ ID NO: 2.
4. The method of claim 3, wherein the HNP-1 polypeptide comprises
an arginine residue that is susceptible to ADP-ribosylation at
amino acid position 14, 24, or a combination thereof.
5. The method of claim 1, wherein the defensin polypeptide is
administered by inhalation.
6. The method of claim 1, further comprising administering to the
subject a therapeutically effective amount of an arginine-specific
mono-ADP-ribosyltransferase (ART) polypeptide.
7. The method of claim 6, wherein the ART polypeptide is ART1 or
ART5.
8. The method of claim 1, wherein the pulmonary disease is cystic
fibrosis, emphysema, asthma, sarcoidosis, chronic bronchitis,
bronchopulmonary dysplasia, pulmonary fibrosis, pneumonia, or adult
respiratory distress syndrome.
9. An in vitro method of producing a polypeptide with altered
activity, comprising: contacting a polypeptide comprising at least
one arginine residue susceptible to ADP-ribosylation with NAD and
an arginine-specific mono-ADP-ribosyltransferase to produce a
polypeptide comprising at least one arginine residue that is
ADP-ribosylated; incubating the ADP-ribosylated polypeptide under
conditions sufficient for conversion of the at least one
ADP-ribosylated arginine residue to ornithine; and isolating the
ornithine-containing polypeptide, thereby producing the polypeptide
with altered activity.
10. The method of claim 9, wherein the polypeptide comprises a
defensin polypeptide.
11. The method of claim 10, wherein the defensin polypeptide
comprises a human neutrophil peptide-1 (HNP-1) polypeptide.
12. The method of claim 11, wherein the HNP-1 polypeptide comprises
the amino acid sequence of SEQ ID NO: 2.
13. The method of claim 12, wherein the ornithine-containing HNP-1
polypeptide comprises ornithine at amino acid position 14, 24, or a
combination thereof.
14. The method of claim 9, wherein the conditions sufficient for
conversion of the at least one ADP-ribosylated arginine residue to
ornithine comprise: (i) incubating the ADP-ribosylated defensin
polypeptide at approximately pH 7 to pH 9; (ii) incubating the
ADP-ribosylated defensin polypeptide at about 30.degree. C. to
37.degree. C.; (iii) incubating the ADP-ribosylated defensin
polypeptide for about 4 to 24 hours; (iv) incubating the
ADP-ribosylated defensin polypeptide at about pH 9 for about 24
hours at about 30.degree. C.; or (v) any combination of (i) to
(iv).
15. The method of claim 9, further comprising administering the
polypeptide with modified activity to a subject with pulmonary
disease.
16. A pharmaceutical composition comprising a therapeutically
effective amount of a defensin polypeptide and NAD, wherein the
defensin polypeptide comprises at least one arginine residue that
is susceptible to ADP-ribosylation.
17. The pharmaceutical composition of claim 16, wherein the
defensin polypeptide comprises human neutrophil peptide-1
(HNP-1).
18. The pharmaceutical composition of claim 17, wherein the HNP-1
comprises the amino acid sequence of SEQ ID NO: 2.
19. The pharmaceutical composition of claim 16, further comprising
a therapeutically effective amount of an arginine-specific
mono-ADP-ribosyltransferase (ART) polypeptide.
20. The pharmaceutical composition of claim 19, wherein the ART
polypeptide is ART1 or ART5.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional of U.S. application Ser. No.
13/394,393, filed Mar. 6, 2012, which is the U.S. National Stage of
International Application No. PCT/US2010/048068, filed Sep. 8,
2010, published in English under PCT Article 21(2), which claims
the benefit of U.S. Provisional Application No. 61/241,311, filed
on Sep. 10, 2009. All of the above-referenced applications are
herein incorporated by reference in their entirety.
FIELD
[0002] This disclosure relates to methods of treating pulmonary
disease, and in vitro methods of producing modified proteins, such
as defensins, of use in treating pulmonary disease.
BACKGROUND
[0003] Mono-ADP-ribosylation is a post-translational modification
of proteins in which the ADP-ribose moiety of nicotinamide adenine
dinucleotide (NAD) is transferred to a specific amino acid. Several
well-characterized mono-ADP-ribosyltransferases have been
identified in viruses, bacteria and eukaryotes (Corda and
DiGirolamo, EMBO J. 22:1953-1958, 2003). Arginine-specific
mono-ADP-ribosyltransferase-1 (ART1) is present on the apical
surface of epithelial cells in human airways and is linked to the
cell surface by a glycosylphosphatidylinositol (GPI) anchor
(Balducci et al., Am. J. Respir. Cell. Mol. Biol. 21:337-346, 1999;
Okazaki and Moss, J. Biol. Chem. 273:23617-23620, 1998). ART1
modifies the arginines of several substrates, including defensins
such as human neutrophil peptide (HNP)-1, thereby altering their
activity (Corda and DiGirolamo Sci. STKE PE53, 2002; DiGirolamo et
al., FEBS J. 272:4565-4575, 2005).
[0004] Neutrophils, a critical component of the innate immune
system, are recruited to airways in response to inflammation or
infection. Neutrophil defensins (HNP-1 to -3), stored in
azurophilic granules, are small cationic peptides whose main
function is to defend the lung against pathogenic micro-organisms
(Bals and Hiemstra, Eur. Respir. J. 23:327-333, 2004). High levels
of defensins have been found in patients with inflammatory lung
diseases such as idiopathic pulmonary fibrosis (IPF) (Mukae et al.,
Thorax 57:623-628, 2002) and cystic fibrosis (Soong et al.,
Inflamm. Res. 46:98-102, 1997). In addition to antimicrobial
activities and other diverse functions (Rehaume and Hancock, Crit.
Rev. Immunol. 28:185-200, 2008), defensins interact with airway
epithelial cells, increasing proliferation and stimulating wound
repair (van Wetering et al., J. Leukoc. Biol. 77:444-450, 2005).
HNPs 1-3 are arginine rich and differ in sequence by one amino
acid. The arginines in HNP-1 are critical for maintaining activity
of the protein (Zou et al, J. Biol. Chem. 282:19653-19665,
2007).
[0005] In vitro, ART1 ADP-ribosylates HNP-1 on arginine 14 with a
secondary site on arginine 24. Mono- and di-ADP-ribosylated HNP-1
have previously been isolated from the bronchoalveolar lavage fluid
(BALF) of IPF and asthma patients, consistent with a role for the
modified HNP-1 in disease (Paone et al., J. Biol. Chem.
281:17054-17060, 2006).
SUMMARY
[0006] There are a number of pulmonary diseases wherein therapeutic
proteins are of use. This disclosure relates to polypeptides of use
in treating those diseases. For example, defensins exhibit a wide
range of antimicrobial and immune stimulatory activities, including
cytotoxicity towards bacterial cells; however, these proteins are
also cytotoxic for mammalian cells, including human epithelial and
endothelial cells. This side effect may limit their usefulness for
treating pulmonary disease. Thus, there exists a need to identify
new methods of modifying existing agents, such as defensins, in
order to modify their cytotoxic activity and potentially improve
their effectiveness for treating pulmonary disease.
[0007] This disclosure provides 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 particular examples,
the polypeptide and/or NAD are administered by inhalation. In
additional examples, the method further includes administering a
therapeutically effective amount of an ART (such as an
arginine-specific ART, for example mammalian ART1, ART2, or ART5).
In some examples, the polypeptide is a defensin, for example, an
alpha defensin, such as human neutrophil peptide-1 (HNP-1). Also
disclosed is a pharmaceutical composition including at least one
polypeptide (such as a defensin) and NAD.
[0008] The disclosure also provides in vitro methods of producing a
polypeptide with altered activity, including contacting a
polypeptide that includes at least one arginine residue susceptible
to ADP-ribosylation with NAD and an arginine-specific
mono-ADP-ribosyltransferase (for example, ART1) to produce a
polypeptide including at least one ADP-ribosylated arginine
residue. The methods include 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. In particular examples, the
conditions sufficient for conversion of ADP-ribosylated arginine to
ornithine include incubation in a solution having a pH of about 7
to 9, incubation for a time of about 4 to 24 hours, and/or
incubation at a temperature of about 30.degree. C. to about
37.degree. C. In some examples, the polypeptide is a defensin, such
as an alpha defensin, such as HNP-1. Polypeptides made using these
methods can be used in the therapeutic methods provided herein.
[0009] Also disclosed are methods of treating a subject with
pulmonary disease, including administering to the subject a
therapeutically effective amount of a modified polypeptide
including at least one ornithine residue in place of an arginine
residue. In particular examples, the modified polypeptide is a
defensin, for example, an alpha defensin, such as HNP-1.
[0010] The foregoing and other features of the disclosure will
become more apparent from the following detailed description, which
proceeds with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a series of chromatograms showing reverse
phase-high performance liquid chromatography (RP-HPLC) separation
of reaction products from incubation of 3 nmol HNP-1 and 5 mM NAD
with the indicated amounts of ART1 at 30.degree. C. overnight.
Peaks were identified by mass spectrometry (MS) as o,
di-ADP-ribosylated-HNP-1; *, ADP-ribosyl-HNP-1-ornithine; +,
mono-ADP-ribosylated-HNP-1; and , HNP-1. Data are representative of
three experiments.
[0012] FIG. 2A is a chromatogram showing RP-HPLC separation of 1
nmol purified ADP-ribosyl-HNP-1-ornithine produced by incubating 10
nmol HNP-1 and 5 mM NAD with 12.8 nmol/h ART1 at 30.degree. C.
overnight, followed by acid hydrolysis. * indicates expected
position of derivatized ornithine.
[0013] FIG. 2B is a chromatogram showing RP-HPLC separation of 3
nmol purified HNP-1 with 23 pmol ornithine added after hydrolysis.
* indicates ornithine.
[0014] FIG. 2C is an RP-HPLC chromatogram of 3 nmol purified
HNP-1.
[0015] FIG. 2D is a MS/MS profile of a tryptic digest of the
ADP-ribosyl-HNP-1-ornithine from FIG. 2A. b8, HNP-1 amino acids 6
(Ile) through 13 (Glu); b9, HNP-1 amino acids 6 (Ile) through 14
(Arg converted to Orn).
[0016] FIG. 2E is a MS/MS profile of a tryptic digest of the HNP-1
from FIG. 2C. b8, HNP-1 amino acids 6 (Ile) through 13 (Glu); b9,
HNP-1 amino acids 6 (Ile) through 14 (Arg).
[0017] FIG. 3 is a graph showing percent of
ADP-ribosyl-HNP-1-ornithine as a function of time in a reaction
containing 3 nmol HNP-1, 5 mM NAD and 5.8 nmol/h ART1 at pH 7.5 and
30.degree. C. ADP-ribosyl-HNP-1-ornithine was quantified as pmoles
calculated from the area (mAu) under the peak identified as
ADP-ribosyl-HNP-1-ornithine at 280 nm. The reported percent was
based on the total number of pmoles of reaction products in the
separation. Data are mean.+-.SD from four experiments.
[0018] FIG. 4A is an HPLC chromatogram of RP-HPLC separation of
reaction products from overnight incubation of 10 nmol HNP-1 and 5
mM NAD with 12.8 nmol/h ART1 at 30.degree. C. Peaks are identified
as .largecircle., di-ADP-ribosylated-HNP-1; *,
ADP-ribosyl-HNP-1-ornithine; +, mono-ADP-ribosylated-HNP-1; and ,
HNP-1.
[0019] FIG. 4B is an HPLC chromatogram of purified
di-ADP-ribosylated HNP-1 at pH 9 (time 0). Data is representative
of three experiments.
[0020] FIG. 4C is an HPLC chromatogram of purified
di-ADP-ribosylated HNP-1 incubated at pH 7 and 30.degree. C. for 24
hours. Data is representative of two experiments.
[0021] FIG. 4D is an HPLC chromatogram of purified
di-ADP-ribosylated HNP-1 incubated at pH 9 and 30.degree. C. for 24
hours. Data is representative of three experiments.
[0022] FIG. 4E is an HPLC chromatogram of purified
mono-ADP-ribosylated HNP-1 at pH 9 (time 0). Data is representative
of three experiments.
[0023] FIG. 4F is an HPLC chromatogram of purified
mono-ADP-ribosylated HNP-1 incubated at pH 7 and 30.degree. C. for
24 hours. Data is representative of two experiments.
[0024] FIG. 4G is an HPLC chromatogram of purified
mono-ADP-ribosylated HNP-1 incubated at pH 9 and 30.degree. C. for
24 hours. Data is representative of three experiments.
[0025] FIG. 5A is an HPLC chromatogram of ADP-ribose and arginine
incubated for 24 hours at 37.degree. C. in 20 mM potassium
phosphate, adjusted to pH 9 by NaOH.
[0026] FIG. 5B is an HPLC chromatogram of ornithine incubated for
24 hours at 37.degree. C. in 20 mM potassium phosphate, adjusted to
pH 9 by NaOH.
[0027] FIG. 5C is an HPLC chromatogram of
ADP-ribosyl-[.sup.14C]arginine incubated for 24 hours at 37.degree.
C. in 6 N HCl.
[0028] FIG. 5D is an HPLC chromatogram of
ADP-ribosyl-[.sup.14C]arginine at time 0.
[0029] FIG. 5E is an HPLC chromatogram of
ADP-ribosyl-[.sup.14C]arginine incubated for 24 hours at 37.degree.
C. in 20 mM potassium phosphate, adjusted to pH 9 by NaOH.
[0030] FIG. 5F is an amino acid analysis monitored by fluorescence
of ADP-ribosyl-[.sup.14C]arginine at time 0. .largecircle.,
ADP-ribosyl-[.sup.14C]arginine; , arginine.
[0031] FIG. 5G is an amino acid analysis monitored by fluorescence
of ornithine (25 pmol). *, ornithine.
[0032] FIG. 5H is an amino acid analysis of
ADP-ribosyl-[.sup.14C]arginine incubated for 24 hours at 37.degree.
C. in 20 mM potassium phosphate, adjusted to pH 9 by NaOH.
.largecircle., ADP-ribosyl-[.sup.14C]arginine; , arginine; *,
ornithine.
SEQUENCE LISTING
[0033] The amino acid sequences listed in the accompanying sequence
listing are shown using standard three letter code for amino acids,
as defined in 37 C.F.R. 1.822.
[0034] The Sequence Listing is submitted as an ASCII text file,
created on Jul. 9, 2014, 7.92 KB, which is incorporated by
reference herein. In the accompanying sequence listing:
[0035] SEQ ID NO: 1 is the amino acid sequence of the HNP-1 and
HNP-2 prepro-protein.
[0036] SEQ ID NO: 2 is the amino acid sequence of HNP-1.
[0037] SEQ ID NO: 3 is the amino acid sequence of HNP-2.
[0038] SEQ ID NO: 4 is the amino acid sequence of the HNP-3
prepro-protein.
[0039] SEQ ID NO: 5 is the amino acid sequence of HNP-3.
[0040] SEQ ID NO: 6 is the amino acid sequence of the HNP-4
prepro-protein.
[0041] SEQ ID NO: 7 is the amino acid sequence of HNP-4.
[0042] SEQ ID NO: 8 is the amino acid sequence of the HD-5
prepro-protein.
[0043] SEQ ID NO: 9 is the amino acid sequence of HD-5.
[0044] SEQ ID NO: 10 is the amino acid sequence of the HD-6
prepro-protein.
[0045] SEQ ID NO: 11 is the amino acid sequence of HD-6.
[0046] SEQ ID NO: 12 is the amino acid sequence of the Def-X
prepro-protein
[0047] SEQ ID NO: 13 is the amino acid sequence of Def-X.
DETAILED DESCRIPTION
[0048] Targeted ADP-ribosylation of specific arginines by ARTs, and
their subsequent replacement with ornithine, is a novel alternative
pathway for regulation of protein function through
post-translational modification. Without being bound by theory, in
addition to altering the molecular charge, secondary structure, and
biological activity, the presence of ornithine at the position of
one or more arginine residues and the absence of ADP-ribose would
prevent a modified protein from interacting with
ADP-ribosylacceptor hydrolases or serving as a target for
subsequent ADP-ribosylation. ADP-ribosylacceptor hydrolases
regulate cellular levels of ADP-ribosylated proteins. Moreover, the
effects of ADP-ribosylation, such as on signal transduction by a
polypeptide, would be altered.
[0049] Defensin polypeptides are antimicrobial peptides that are
involved in the innate immune defense and are cytotoxic for
microbes such as bacteria, fungi, and certain types of viruses. In
addition, they stimulate IL-8 release from neighboring cells and
induce an increase in T cell chemotaxis. The ADP-ribosylation of an
arginine residue in an HNP-1 defensin polypeptide can alter its
antimicrobial activity and/or modify an immune response (e.g., U.S.
Pat. No. 7,511,015).
[0050] It is shown herein that ADP-ribosylated arginine residues in
a polypeptide, such as a defensin polypeptide, are converted
non-enzymatically to ornithine in vitro. Conversion of
ADP-ribosylated arginine to ornithine also occurs in vivo, as
demonstrated by the presence of HNP-1 containing both ADP-ribose
and ornithine in bronchoalveolar lavage fluid from a patient with
idiopathic pulmonary fibrosis.
[0051] Administering a polypeptide (such as a defensin) with at
least one arginine susceptible to ADP-ribosylation and NAD to a
subject (for example, by inhalation) provides the substrate and
ADP-ribose source for production of ADP-ribosylated polypeptide
because of the presence of ADP-ribosyltransferases on the surface
of epithelial cells and inflammatory cells or secreted into the
airway lumen. Subsequent conversion of the ADP-ribosylated arginine
to ornithine can occur, which can be useful for treating pulmonary
disease. Similarly, a polypeptide (such as a defensin) in which at
least one arginine residue is replaced with an ornithine can be
produced in vitro as described herein. The modified
ornithine-containing polypeptide can be administered to a subject
for treating pulmonary disease.
I. Abbreviations
[0052] ADP: adenosine diphosphate
[0053] ART1: arginine-specific mono-ADP-ribosyltransferase-1
[0054] BALF: bronchoalveolar lavage fluid
[0055] HNP: human neutrophil peptide
[0056] HPLC: high-performance liquid chromatography
[0057] IPF: idiopathic pulmonary fibrosis
[0058] MS: mass spectrometry
[0059] NAD: nicotinamide adenine dinucleotide
[0060] OPA: o-phthalaldehyde
[0061] Orn: ornithine
[0062] RP-HPLC: reverse phase high-performance liquid
chromatography
II. Terms
[0063] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes V, published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
[0064] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this disclosure belongs.
The singular terms "a," "an," and "the" include plural referents
unless context clearly indicates otherwise. Similarly, the word
"or" is intended to include "and" unless the context clearly
indicates otherwise. Hence "comprising A or B" means including A,
or B, or A and B. It is further to be understood that all base
sizes or amino acid sizes, and all molecular weight or molecular
mass values, given for nucleic acids or polypeptides are
approximate, and are provided for description. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of the present disclosure, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. All GenBank Accession
numbers mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including
explanations of terms, will control. In addition, the materials,
methods, and examples are illustrative only and not intended to be
limiting.
[0065] In order to facilitate review of the various embodiments of
the disclosure, the following explanations of specific terms are
provided:
[0066] Administration: Administration of an active compound
("agent") or composition (such as those peptides provided herein)
can be by any route known to one of skill in the art.
Administration can be local or systemic. Examples of local
administration include, but are not limited to, topical
administration, subcutaneous administration, intramuscular
administration, intrathecal administration, intrapericardial
administration, intra-ocular administration, topical ophthalmic
administration, or administration to the nasal mucosa or lungs by
inhalational administration. In addition, local administration
includes routes of administration typically used for systemic
administration, for example by directing intravascular
administration to the arterial supply for a particular organ.
Systemic administration includes any route of administration
designed to distribute an active compound or composition widely
throughout the body via the circulatory system. Thus, systemic
administration includes, but is not limited to intra-arterial and
intravenous administration. Systemic administration also includes,
but is not limited to, topical administration, subcutaneous
administration, intramuscular administration, or administration by
inhalation, when such administration is directed at absorption and
distribution throughout the body by the circulatory system.
[0067] ADP-ribosylation: Reaction in which ADP-ribose is covalently
attached to another compound. A family of eukaryotic and
prokaryotic mono-ADP-ribosyltransferases (ARTs) catalyze the
transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD)
to proteins.
[0068] As disclosed herein, a number of proteins used in host
defense are basic and arginine-rich and thus could serve as
acceptors for ADP-ribose. These include, but may not be limited to,
alpha defensins (HNP-1, HNP-2, HNP-3, HNP-4, HD-5, HD-6); beta
defensins (hBD1, hBD-2, hBD-3, hBD-4); Major Basic Protein;
Eosinophil Cationic Protein; cathelicidin antimicrobial peptide
(e.g., hCAP18) and lysozyme.
[0069] Altered activity: As used herein, an ornithine-containing
polypeptide with "altered activity" refers to any polypeptide with
at least one ornithine residue that exhibits a change in its
ability to interact with another molecule (such as an increase or
decrease in binding affinity to another molecule) or exhibits a
change (such as an increase or decrease) in one or more functions
or biological activities of the protein. In some examples, when the
ornithine-containing polypeptide is a defensin, an altered activity
can be a change in the polypeptide's antimicrobial activity. An
alteration in antimicrobial activity can include, for example, an
increase in cytokine production or an increase in recruitment of
inflammatory cells, as discussed in greater detail in the sections
below.
[0070] Arginine: An amino acid (C.sub.6H.sub.14N.sub.4O.sub.2)
found in plants and animals that is essential for the human diet;
also produced by the breakdown of proteins. Also encompassed are
functional analogues of arginine, and structurally modified
arginine molecules (e.g., agmatine).
[0071] Defensins: Small, cationic peptides that have six conserved
cysteine residues that form three disulfide bonds. Functional
defensins arise by the sequential post-translational processing of
a prepro-protein of 93-95 amino acids in length. The members of the
defensin family are divided into different classes. The
alpha-defensins generally contain 29-33 residues. The
beta-defensins are more basic than alpha defensins and are
generally between 34-37 amino acids in length. See, e.g., Raj et
al., Biochem J.; 347:633-41, 2000. The recently identified theta
defensins are formed by the head-to-tail linkage of two alpha
defensin-related nonapeptides, generating a circular 18-residue
polypeptide (Tang et al., Science; 286:498-502, 1999).
[0072] Defensins were first identified in neutrophils and have been
detected in human, rabbit, guinea pig, and rat phagocytes. Alpha
defensins include, but are not limited to, HNP-1, HNP-2, HNP-3,
HNP-4, human defensin (HD)-5, and HD-6. Alpha defensins also
include the recently identified HNP-4 homolog, defensin (Def)-X
(see U.S. Pat. No. 6,329,340).
[0073] HNP-1 and HNP-2 are products of the same gene (GenBank
Accession No. NP.sub.--004075 herein incorporated by reference as
present in GenBank on Sep. 10, 2009). HNP-3 differs from HNP-1 by
only one amino acid, but is the product of a different
prepro-protein (GenBank Accession No. NP.sub.--005208, herein
incorporated by reference as present in GenBank on Sep. 10, 2009).
HNP-4 is the product of a different gene (GenBank Accession No.
NP.sub.--001916 herein incorporated by reference as present in
GenBank on Sep. 10, 2009). HD-5 (GenBank Accession No.
NP.sub.--066290) and HD-6 (GenBank Accession No. NP.sub.--001917),
each herein incorporated by reference as present in GenBank on Sep.
10, 2009, are two human enteric defensins.
[0074] Defensins are toxic for a variety of infectious agents, such
as Gram-negative bacteria, Gram-positive bacteria, fungi, and
certain enveloped viruses. Defensins act by forming pores in
membranes of the infectious agent and generating voltage-dependent
channels. Antimicrobial activities of defensins include, but are
not limited to, lysis of bacteria, fungi, or viruses; toxicity for
bacteria, fungi or viruses; leukocyte (e.g., T cell) chemotaxis;
and leukocyte (e.g., neutrophil) recruitment. Without being bound
by theory, defensins play an important role in the body's natural
immunity against infections. Defensins are also cytotoxic for
several normal and malignant cells. A "modified defensin" is a
defensin that includes at least one modified arginine residue (such
as an ADP-ribosylated arginine) or a defensin that contains at
least one ornithine residue in place of an arginine residue. An
"unmodified defensin" is a defensin that includes only unmodified
(native) arginine residues.
[0075] Isolated: A biological component (such as a nucleic acid,
peptide or protein) that has been substantially separated, produced
apart from, or purified away from other biological components in
the cell of the organism in which the component naturally occurs,
e.g., other chromosomal and extrachromosomal DNA and RNA, and
proteins. Nucleic acids, peptides and proteins that have been
"isolated" thus include nucleic acids and proteins purified by
standard purification methods. The term also embraces nucleic
acids, peptides and proteins prepared by recombinant expression in
a host cell, as well as chemically synthesized nucleic acids and
proteins.
[0076] Mono-ADP-ribosyltransferase (ART): A family of enzymes that
catalyze the transfer of ADP-ribose from NAD to proteins. Mammalian
ARTs that are secreted or localized on the cell surface through
glycosylphosphatidylinositol (GPI) anchors are expressed
preferentially by epithelial and inflammatory cells such as
lymphocytes and neutrophils. Substrates of the five known mammalian
ADP-ribosyltransferases (ART1, ART2, ART3, ART4, and ART5) include
proteins that are involved in critical cellular events (e.g.,
lymphocyte activation, neutrophil chemotaxis). Three of these
transferases, ART1, ART2, and ART5, specifically modify arginine
residues in proteins.
[0077] Bacterial products (e.g., cholera toxin, pertussis toxin,
and diphtheria toxin) are also included among the ARTs.
[0078] Nicotinamide adenine dinucleotide (NAD): A dinucleotide
compound containing an adenine base and a nicotinamide joined
through their phosphate groups having the structure:
##STR00001##
[0079] NAD is an essential enzyme cofactor in metabolism. For
example, it acts as a coenzyme in redox reactions, providing
transfer of electrons from one redox reaction to another. It also
serves as a donor of ADP-ribose groups in ADP-ribosylation
reactions and as a precursor of the second messenger molecule
cyclic ADP-ribose.
[0080] Ornithine: An amino acid (C.sub.5H.sub.12N.sub.2O.sub.2)
which is part of the urea cycle having the structure:
##STR00002##
Ornithine and urea are produced by the hydrolysis of arginine,
which is catalyzed by arginase. Ornithine may also be produced
non-enzymatically from ADP-ribosylated arginine. Ornithine is not
coded for by DNA, and is therefore not incorporated into naturally
occurring primary amino acid sequences.
[0081] Polypeptide: A polymer in which the monomers are amino acid
residues that are joined together through amide bonds. When the
amino acids are alpha-amino acids, either the L-optical isomer or
the D-optical isomer can be used, the L-isomers being preferred in
nature. The term polypeptide or protein as used herein encompasses
any amino acid sequence and includes, but may not be limited to,
modified sequences such as ADP-ribosylated proteins,
ribosyl-proteins, ornithine-containing proteins, and glycoproteins.
The term polypeptide is specifically intended to cover naturally
occurring proteins, as well as those that are recombinantly or
synthetically produced.
[0082] Polypeptides (such as those provided herein) may include one
or more conservative amino acid substitutions. Conservative
substitutions are the substitution of an amino acid residue for
another amino acid residue having similar biochemical properties.
Typically, conservative substitutions have little to no impact on
the biological activity of a resulting polypeptide. In a particular
example, a conservative substitution is an amino acid substitution
in a peptide that does not substantially affect the biological
function of the peptide. A peptide can include one or more amino
acid substitutions, for example 1-10 conservative substitutions,
2-5 conservative substitutions, or 4-9 conservative
substitutions.
[0083] Conservative amino acid substitution tables providing
functionally similar amino acids are well known to one of ordinary
skill in the art. The following six groups are examples of amino
acids that are considered to be conservative substitutions for one
another:
[0084] 1) Alanine (A), Serine (S), Threonine (T);
[0085] 2) Aspartic acid (D), Glutamic acid (E);
[0086] 3) Asparagine (N), Glutamine (Q);
[0087] 4) Arginine (R), Lysine (K);
[0088] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
and
[0089] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0090] Any polypeptide sequence variant will preferably introduce
no more than twenty, and preferably fewer than ten amino acid
substitutions (for example, conservative substitutions) into the
encoded polypeptide. Thus, the disclosed peptide, such as the
disclosed defensins, can have 1-10, 1-5, or 1, 2, 3, 4, or 5
conservative amino acid substitutions shown above. Variant amino
acid sequences may, for example, be 80%, 90%, 95%, 98%, 99%, or
more identical to the native amino acid sequence. Programs and
algorithms for determining percentage identity can be found at the
NCBI website.
[0091] Pharmaceutical agent or composition: A chemical compound or
composition capable of inducing a desired therapeutic or
prophylactic effect when properly administered to a subject or a
cell.
[0092] Pharmaceutically acceptable carriers: The pharmaceutically
acceptable carriers useful in this disclosure are conventional.
Remington: The Science and Practice of Pharmacy, The University of
the Sciences in Philadelphia, Editor, Lippincott, Williams, &
Wilkins, Philadelphia, Pa., 21.sup.st Edition (2005), describes
compositions and formulations suitable for pharmaceutical delivery
of compounds, such as peptides or modified peptides, such as alpha
defensins or modified alpha defensins.
[0093] In general, the nature of the carrier will depend on the
particular mode of administration employed. For instance,
parenteral formulations usually comprise injectable fluids that
include pharmaceutically and physiologically acceptable fluids such
as water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol, or the like as a vehicle. For solid
compositions (e.g., powder, pill, tablet, or capsule forms),
conventional non-toxic solid carriers can include, for example,
pharmaceutical grades of mannitol, lactose, starch, or magnesium
stearate. In addition to biologically-neutral carriers,
pharmaceutical compositions to be administered can contain minor
amounts of non-toxic auxiliary substances, such as wetting or
emulsifying agents, preservatives, pH buffering agents, or the
like, for example sodium acetate or sorbitan monolaurate.
[0094] Pulmonary disease: A disease of the respiratory system,
including the lungs and bronchial tree. Pulmonary diseases include,
for example, cystic fibrosis, emphysema, asthma, sarcoidosis,
chronic bronchitis, bronchopulmonary dysplasia, pulmonary fibrosis,
pneumonia, and adult respiratory distress syndrome.
[0095] Purified: The term purified does not require absolute
purity; rather, it is intended as a relative term. Thus, for
example, a purified peptide preparation is one in which the peptide
or protein is more enriched than the peptide or protein is in its
natural environment within a cell. Preferably, a preparation is
purified such that the protein or peptide represents at least 50%
of the total peptide or protein content of the preparation.
[0096] Substantially purified polypeptide as used herein refers to
a polypeptide (such as a defensin polypeptide, for example a
modified defensin polypeptide) that is substantially free of other
proteins, lipids, carbohydrates or other materials with which it is
naturally associated. In one embodiment, the polypeptide is at
least 50%, for example at least 80%, free of other proteins,
lipids, carbohydrates or other materials with which it is naturally
associated. In another embodiment, the polypeptide is at least 90%
free of other proteins, lipids, carbohydrates or other materials
with which it is naturally associated. In yet another embodiment,
the polypeptide is at least 95% free of other proteins, lipids,
carbohydrates or other materials with which it is naturally
associated.
[0097] Recombinant: A recombinant nucleic acid or protein is one
that has a sequence that is not naturally occurring or was made
artificially. Artificial combination is often accomplished by
chemical synthesis or, more commonly, by the artificial
manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques. In some cases, a recombinant
protein is one encoded by a recombinant nucleic acid molecule.
[0098] Subject: Living multi-cellular vertebrate organisms, a
category that includes both human and non-human mammals. Includes
subjects that have or may be susceptible to pulmonary disease.
[0099] Susceptible to ADP-ribosylation: A molecule (for example an
amino acid residue, such as an arginine residue) that can be
modified by an ADP-ribose (is available for ADP-ribosylation).
ADP-ribosylation of a molecule (such as an arginine residue) can be
determined by one of skill in the art for example, using the
methods described herein, such as HPLC or MS methods. Such methods
can identify which residue(s) include the ADP-ribose modification,
thus identifying the residue(s) that are susceptible to
ADP-ribosylation. In a particular example, the amino acid arginine
(either isolated arginine, or arginine present in a polypeptide or
protein, such as a defensin) can be modified by an ADP-ribose, for
example, by transfer of an ADP-ribose to arginine to produce
ADP-ribosyl-arginine, such as by an ADP-ribosyltransferase.
[0100] Therapeutically effective amount: An amount or dose
sufficient to prevent advancement, or to cause regression of a
disease (such as pulmonary disease, for example, cystic fibrosis,
emphysema, asthma, sarcoidosis, chronic bronchitis,
bronchopulmonary dysplasia, pulmonary fibrosis, pneumonia, or adult
respiratory distress syndrome), or which is capable of relieving
symptoms caused by the disease.
[0101] Treatment: Refers to both prophylactic inhibition of initial
infection or disease, and therapeutic interventions to alter the
natural course of an untreated infection or disease process, such
as a pulmonary disease.
III. Methods of Treating Pulmonary Disease
[0102] Disclosed herein are methods of treating a subject with
pulmonary disease. In some examples, the method includes
administering to the subject a therapeutically effective amount of
a polypeptide with at least one arginine residue susceptible to
ADP-ribosylation and NAD. In some embodiments, the polypeptide
and/or NAD are administered by inhalation. In some examples, the
polypeptide is susceptible to ADP-ribosylation on more than one
arginine residue simultaneously such as, at least two, at least
three, at least four, or more arginine residues. In additional
examples, the method also includes administering a therapeutically
effective amount of an ART (such as ART1) to the subject.
[0103] In some examples, the polypeptide includes an enzyme, a
cytokine, a chemokine, an anti-inflammatory peptide, a receptor
ligand, a hormone, an antigen, or an antibody. In particular
examples, the polypeptide is a defensin, such as an alpha defensin
polypeptide (for example a mammalian alpha defensin, such as a
human, monkey, rabbit, rat, cat, dog, pig, sheep, or mouse alpha
defensin). In a specific, non-limiting example, the alpha defensin
is human neutrophil peptide (HNP)-1. In other specific,
non-limiting examples, the alpha defensin polypeptide is HNP-2,
HNP-3, HNP-4, HD-5, HD-6, or Def-X.
[0104] The alpha defensins include HNP-1, HNP-2, HNP-3, HNP-4,
HD-5, HD-6, and Def-X. HNP-1 and HNP-2 are products of the same 94
amino acid prepro-protein. In one embodiment, this protein has the
following sequence:
TABLE-US-00001 MRTLAILAAILLVALQAQAEPLQARADEVAAAPEQIAADIPEVVVSL
AWDESLAPKHPGSRKNMACYCRIPACIAGERRYGTCIYQGRLWAFCC; (SEQ ID NO: 1, see
also GenBank Accession No. NP_004075, herein incorporated by
reference as present in GenBank on Sep. 10, 2009).
[0105] HNP-1 is one member of the family of alpha defensins
produced by cleavage of the prepro-protein. In one embodiment,
HNP-1 has a sequence as set forth as:
TABLE-US-00002 (SEQ ID NO: 2) ACYCRIPACIAGERRYGTCIYQGRLWAFCC;.
[0106] HNP-2 is another member of the family of alpha defensins
produced by cleavage of the prepro-protein. In one embodiment,
HNP-2 has a sequence as set forth as:
TABLE-US-00003 (SEQ ID NO: 3) CYCRIPACIAGERRYGTCIYQGRLWAFCC;.
[0107] HNP-3 is a third member of the family of alpha defensins
produced by cleavage of a prepro-protein. In one embodiment, the
HNP-3 prepro-protein has a sequence set forth as:
TABLE-US-00004 MRTLAILAAILLVALQAQAEPLQARADEVAAAPEQIAADIPEVVVSL
AWDESLAPKHPGSRKNMDCYCRIPACIAGERRYGTCIYQGRLWAFCC (SEQ ID NO: 4, see
also, GenBank Accession No. NP_005208, herein incorporated by
reference as present in GenBank on Sep. 10, 2009).
[0108] In one embodiment, HNP-3 has a sequence as set forth as:
TABLE-US-00005 (SEQ ID NO: 5) DCYCRIPACIAGERRYGTCIYQGRLWAFCC;.
[0109] HNP-4 is an alpha defensin that is the product of a
prepro-protein having a sequence as set forth as:
TABLE-US-00006 MRIIALLAAILLVALQVRAGPLQARGDEAPGQEQRGPEDQDISISFAWD
KSSALQVSGSTRGMVCSCRLVFCRRTELRVGNCLIGGVSFTYCCTRVD (SEQ ID NO: 6, see
also GenBank Accession No. NP_001916, herein incorporated by
reference as present in GenBank on Sep. 10, 2009).
[0110] In one embodiment, HNP-4 has a sequence as set forth as:
TABLE-US-00007 (SEQ ID NO: 7)
VCSCRLVFCRRTELRVGNCLIGGVSFTYCCTRVD;.
[0111] HD-5 is produced by cleavage of the following prepro-protein
having a sequence as set forth as:
TABLE-US-00008 MRTIAILAAILLVALQAQAESLQERADEATTQKQSGEDNQDLAISFA
GNGLSALRTSGSQARATCYCRTGRCATRESLSGVCEISGRLYRLCCR; (SEQ ID NO: 8,
GenBank Accession No. NP_066290, herein incorporated by reference
as present in GenBank on Sep. 10, 2009).
[0112] In one embodiment, HD-5 has a sequence as set forth as:
TABLE-US-00009 (SEQ ID NO: 9)
ATCYCRTGRCATRESLSGVCEISGRLYRLCCR;.
[0113] HD-6 is produced by cleavage of the following prepro-protein
having a sequence as set forth as:
TABLE-US-00010 MRTLTILTAVLLVALQAKAEPLQAEDDPLQAKAYEADAQEQRGANDQ
DFAVSFAEDASSSLRALGSTRAFTCHCRRSCYSTEYSYGTCTVMGIN HRFCCL; (SEQ ID NO:
10, GenBank Accession No. NP_001917, herein incorporated by
reference as present in GenBank on Sep. 10, 2009).
[0114] In one embodiment, HD-6 has a sequence as set forth as:
TABLE-US-00011 (SEQ ID NO: 11) TCHCRRSCYSTEYSYGTCTVMGINHRFCCL;.
[0115] Def-X is produced by cleavage of the following
prepro-protein having a sequence as set forth as:
TABLE-US-00012 MRTLTLLSAFLLVALQAWAEPLQARAHEMPAQKQPPADDQDVVIYFSG
DDSCSLQVPGSTKGLICHCRVLYCIFGEHLGGTCFILGERYPICCY (SEQ ID NO: 12, see
U.S. Pat. No. 6,329,340).
[0116] In one embodiment, Def-X has a sequence as set forth as:
TABLE-US-00013 (SEQ ID NO: 13) ICHCRVLYCIFGEHLGGTCFILGERYPICCY.
[0117] In particular examples, the defensin is HNP-1 (for example,
SEQ ID NO: 2). In some examples, HNP-1 is susceptible to
ADP-ribosylation on an arginine residue at position 14 of SEQ ID
NO: 2. In other examples, HNP-1 is susceptible to ADP-ribosylation
on an arginine residue at position 24 of SEQ ID NO: 2. In some
examples, HNP-1 is susceptible to ADP-ribosylation on more than one
arginine residue simultaneously such as, at least two, at least
three, or at least four arginine residues. In a particular example,
HNP-1 is susceptible to ADP-ribosylation on arginines at both
position 14 and position 24 of SEQ ID NO: 2.
[0118] The disclosed methods include administering a
therapeutically effective amount of a polypeptide including at
least one arginine residue susceptible to ADP-ribosylation to a
subject. An arginine residue susceptible to ADP-ribosylation is an
arginine residue (for example, an arginine residue in a
polypeptide) that can be modified by an ADP-ribose (is available
for ADP-ribosylation), for example, by transfer of an ADP-ribose to
the arginine residue by an ART. ADP-ribosylation of an arginine
residue can be determined by one of skill in the art for example,
using the methods described herein, such as HPLC or MS methods.
Such methods can identify which arginine residue(s) include the
ADP-ribose modification, thus identifying the residue(s) that are
susceptible to ADP-ribosylation.
[0119] A therapeutically effective amount of a polypeptide can be
the amount of the polypeptide necessary to treat a pulmonary
disease of the subject in conjunction with administration of NAD,
for example, by increasing ADP-ribosylation of the polypeptide and
its subsequent conversion to a polypeptide containing at least one
ornithine residue in place of an arginine residue. In some
examples, the polypeptide can be administered in dosages from about
1 .mu.g/kg body weight to about 100 mg/kg body weight (such as
about 1 .mu.g/kg to 1 mg/kg or about 10 .mu.g/kg to 10 mg/kg). The
provided amounts are exemplary doses; one of skill in the art can
readily determine suitable dosages. In some examples, the
polypeptide is administered to the subject via inhalation.
[0120] In particular examples, the polypeptide is a defensin, such
as an alpha defensin, for example, HNP-1. In some examples, HNP-1
is administered to a subject with pulmonary disease in dosages from
about 1 .mu.g/kg body weight to about 100 mg/kg body weight (such
as about 1 .mu.g/kg to 1 mg/kg or about 10 .mu.g/kg to 10
mg/kg).
[0121] The disclosed methods also include administering a
therapeutically effective amount of NAD to the subject via
inhalation. NAD serves as a source of ADP-ribose, which can be
transferred to a polypeptide (such as a defensin, for example,
HNP-1) by an ART, which may be present in the airway on epithelial
cells or on inflammatory cells recruited to the lung in a disease
state. A therapeutically effective amount of NAD can be the amount
of NAD necessary to treat a pulmonary disease of a subject, for
example, by increasing ADP-ribosylation of a defensin polypeptide
and its subsequent conversion to a defensin containing at least one
ornithine residue in place of an arginine residue. In some
examples, the amount of NAD may be about 0.5 .mu.M to 20 mM
achieved surface concentration (for example, about 5 .mu.M to about
2 mM, about 25 .mu.M to about 1 mM, or about 50 .mu.M to about 200
.mu.M achieved surface concentration of NAD). An "achieved surface
concentration" is the concentration of a particular compound, such
as NAD, that is achieved, or present, at the surface of a cell or
tissue, for example, at the surface of lung epithelial lining. The
provided amounts are exemplary doses; one of skill in the art can
readily determine suitable dosages.
[0122] In some examples, the method further includes administering
a therapeutically effective amount of an ART polypeptide to the
subject in addition to the polypeptide (for example, a defensin,
such as HNP-1) and NAD. In some examples, the ART is ART1, ART2, or
ART5. In a particular example, the ART is ART1. A therapeutically
effective amount can be the amount of ART polypeptide necessary to
treat a pulmonary disease of the subject in conjunction with
administration of a polypeptide (such as a defensin, for example,
HNP-1) and NAD, for example, by increasing ADP-ribosylation of the
polypeptide and its subsequent conversion to a polypeptide
containing at least one ornithine residue in place of an arginine
residue. In some examples, the ART polypeptide can be administered
in dosages from about 1 .mu.g/kg body weight to about 100 mg/kg
body weight (such as about 1 .mu.g/kg to 1 mg/kg or about 10
.mu.g/kg to 10 mg/kg). The provided amounts are exemplary doses;
one of skill in the art can readily determine suitable dosages.
[0123] Also disclosed herein is a method of treating a subject with
pulmonary disease including administering a therapeutically
effective amount of a modified polypeptide, wherein the modified
polypeptide includes at least one ornithine residue in place of an
arginine residue (such as a modified polypeptide including at least
one ornithine residue produced in vitro as described in Section IV,
below). In some examples, the modified polypeptide includes a
modified enzyme, cytokine, chemokine, anti-inflammatory peptide,
receptor ligand, hormone, antigen, or antibody. In a particular
example, the modified polypeptide is a modified defensin. In some
examples, the modified polypeptide includes more than one ornithine
residue in place of an arginine residue, such as at least two, at
least three, at least four, or more ornithine residues.
[0124] A therapeutically effective amount of a modified polypeptide
including at least one ornithine residue in place of an arginine
residue can be the amount necessary to treat a pulmonary disease of
the subject. In some examples, the modified polypeptide can be
administered in dosages from about 1 .mu.g/kg body weight to about
100 mg/kg body weight (such as about 1 .mu.g/kg to 1 mg/kg or about
10 .mu.g/kg to 10 mg/kg). The provided amounts are exemplary doses;
one of skill in the art can readily determine suitable dosages.
[0125] In particular examples, the modified polypeptide is a
modified defensin polypeptide, such as a modified HNP-1
polypeptide. In particular, non-limiting examples, the modified
defensin polypeptide is an HNP-1 polypeptide including an ornithine
residue at position 14 of HNP-1 (for example, position 14 of SEQ ID
NO: 2), an HNP-1 polypeptide including an ornithine residue at
position 24 of HNP-1 (for example, position 24 of SEQ ID NO: 2), or
an HNP-1 polypeptide including an ornithine residue at positions 14
and 24 of HNP-1 (for example, positions 14 and 24 of SEQ ID NO: 2).
In some examples, the modified HNP-1 polypeptide can be
administered in dosages from about 1 .mu.g/kg body weight to about
100 mg/kg body weight (such as about 1 .mu.g/kg to 1 mg/kg or about
10 .mu.g/kg to 10 mg/kg).
[0126] The disclosed methods include treating a subject with
pulmonary disease, including, but not limited to, cystic fibrosis,
emphysema, asthma, sarcoidosis, chronic bronchitis,
bronchopulmonary dysplasia, pulmonary fibrosis, pneumonia, and
adult respiratory distress syndrome. The subject can be any mammal.
In one example, the subject is human. In other examples, the
subject may be a monkey, rabbit, rat, mouse, pig, sheep, dog, or
cat.
[0127] Cystic fibrosis is a recessive genetic disease in which the
exocrine glands of afflicted individuals produce abnormally thick
mucus which block the intestines and lung passageways and produce
scarring and lesions in the lungs and pancreas.
[0128] Emphysema is condition in which there is over-inflation of
structures in the lungs known as alveoli, or air sacs. This
over-inflation results from a breakdown of the walls of the
alveoli, which causes a decrease in respiratory function and often,
breathlessness. Early symptoms of emphysema include shortness of
breath and cough. Emphysema (together with chronic bronchitis) is
considered chronic obstructive pulmonary disease (COPD).
[0129] Asthma is a disorder of the respiratory system characterized
by inflammation, narrowing of the airways and increased reactivity
of the airways to inhaled agents. Asthma is frequently, although
not exclusively, associated with atopic or allergic symptoms.
[0130] Sarcoidosis is a disease of unknown cause in which
inflammation, consisting of granulomas (clusters of macrophages,
lymphocytes, and multinucleated giant cells), occurs in lymph
nodes, lungs, liver, eyes, skin, or other tissues. Possible causes
of sarcoidosis include a hypersensitivity response, a genetic
predisposition, infection, or chemicals.
[0131] Chronic bronchitis is an inflammation of the lining of the
bronchial tubes. When the bronchi are inflamed and/or infected,
less air is able to flow to and from the lungs and a heavy mucus or
phlegm is coughed up, resulting in bronchitis. A brief attack of
acute bronchitis with cough and mucus production can occur with
severe colds. Chronic bronchitis is characterized by the presence
of a mucus-producing cough most days of the month, three months of
a year for two successive years without other underlying disease to
explain the cough. It may precede or accompany pulmonary emphysema.
Cigarette smoking is by far the most common cause of chronic
bronchitis. The bronchial tubes of people with chronic bronchitis
may also have been irritated initially by bacterial or viral
infections. Air pollution and industrial dusts and fumes are also
causes. Once the bronchial tubes have been irritated over a long
period of time, excessive mucus is produced constantly, the lining
of the bronchial tubes becomes thickened, an irritating cough
develops, air flow may be hampered, and the lungs are endangered.
The bronchial tubes then make an ideal breeding place for
infectious agents. Chronic bronchitis (together with emphysema) is
considered to be COPD.
[0132] Bronchopulmonary dysplasia (BPD) is a chronic lung disorder
that is most common among children who were born prematurely, with
low birth weight and who received prolonged mechanical ventilation
to treat respiratory distress syndrome. BPD is clinically defined
as oxygen dependence to 21 post-natal days. BPD is characterized by
inflammation and scarring in the lungs. More specifically, the high
pressures of oxygen delivery result in necrotizing bronchiolitis
and alveolar septal injury, further compromising oxygenation of
blood. With the advent of surfactant therapy and high frequency
nasal ventilation and oxygen supplementation, infants with BPD
experience much milder injury without necrotizing bronchiolitis or
alveolar septal fibrosis. Instead, there usually are uniformly
dilated acini with thin alveolar septa and little or no
interstitial fibrosis.
[0133] Pulmonary fibrosis (or idiopathic pulmonary fibrosis, IPF)
is a chronic inflammation and progressive fibrosis of alveolar
walls, with steady, progressive shortness of breath, resulting in
death from lack of oxygen or right heart failure. The word
"idiopathic" is used to describe the disease because the cause of
IPF is unknown. Currently, it is believed that IPF may result from
either an autoimmune disorder or the after effects of an infection,
most likely a virus.
[0134] Pneumonia is an inflammation of the lungs caused by a
bacterial, viral, or fungal infection for example, Pneumococcus,
Streptococcus, Hemolyticus, Staphylococcus, Friedlander's bacillus
(Klebsiella pneumonia), and influenza bacillus. Symptoms include
high fever, chest pain, difficulty breathing, coughing and
sputum.
[0135] Adult respiratory distress syndrome (ARDS) is a sudden
pulmonary interstitial and alveolar edema, which usually develops
within a few days after an initiating trauma. ARDS is thought to
result from alveolar injury that has led to increased capillary
permeability. It is also sometimes called acute respiratory
distress syndrome.
[0136] The effectiveness of treatment of a subject with a pulmonary
disease with a polypeptide and NAD (administered with or without an
ART), or with a modified polypeptide containing at least one
ornithine residue, can be measured by assessing the signs and
symptoms of the disease (for example, frequency or severity of
symptoms) and monitoring pulmonary function by methods known to
those of skill in the art. For example, various measurable
parameters of lung function can be studied before, during, or after
treatment. Pulmonary function can be monitored by testing any of
several physically measurable operations of a lung including, but
not limited to, inspiratory flow rate, expiratory flow rate, and
lung volume. An increase (such as a statistically significant
increase), as determined by mathematical formulas well known to
those skilled in the art, in one or more of these parameters
indicates efficacy of the treatment with the polypeptide and NAD
(administered with or without an ART) or the modified polypeptide
containing at least one ornithine residue.
[0137] The methods of measuring pulmonary function most commonly
employed in clinical practice involve timed measurement of
inspiratory and expiratory maneuvers to measure specific
parameters. For example, forced vital capacity (FVC) measures the
total volume in liters exhaled by a patient forcefully from a deep
initial inspiration. This parameter, when evaluated in conjunction
with the forced expiratory volume (FEV1), allows
bronchoconstriction to be quantitatively evaluated. An increase
(such as a statistically significant increase), as determined by
mathematical formulas well known to those skilled in the art, in
FVC or FEV1 reflects a decrease in bronchoconstriction, and
indicates that the polypeptide and NAD therapy (administered with
or without an ART), or therapy with the modified polypeptide
containing at least one ornithine residue is effective (for example
an increase relative to the absence of the therapy with the
modified polypeptide containing at least one ornithine
residue).
[0138] A problem with forced vital capacity determination is that
the forced vital capacity maneuver (forced exhalation from maximum
inspiration to maximum expiration) is largely technique dependent.
In other words, a given subject may produce different FVC values
during a sequence of consecutive FVC maneuvers. The forced
expiratory lung flow (FEF) 25-75 or FEF determined over the
midportion of a forced exhalation maneuver tends to be less
technique dependent than the FVC. Similarly, the FEV1 tends to be
less technique-dependent than FVC. Thus, an increase (such as a
statistically significant increase), as determined by mathematical
formulas well known to those skilled in the art, in the FEF 25-75
or FEV1 reflects a decrease in bronchoconstriction, and indicates
that the polypeptide and NAD therapy (administered with or without
an ART), or therapy with modified polypeptide containing at least
one ornithine residue is effective(for example an increase relative
to the absence of the therapy with the modified polypeptide
containing at least one ornithine residue).
[0139] In addition to measuring volumes of exhaled air as indices
of pulmonary function, the flow in liters per minute measured over
differing portions of the expiratory cycle can be useful in
determining the status of a subject's pulmonary function. In
particular, the peak expiratory flow, taken as the highest airflow
rate in liters per minute during a forced maximal exhalation, is
well correlated with overall pulmonary function in a patient with
asthma and other respiratory diseases. Thus, an increase (such as a
statistically significant increase), as determined by mathematical
formulas well known to those skilled in the art, in the peak
expiratory flow following administration of the polypeptide and NAD
(administered with or without an ART), or administration of the
modified polypeptide containing at least one ornithine residue,
indicates that the therapy is effective (for example an increase
relative to the absence of the therapy with the polypeptide and NAD
(administered with or without an ART) or the modified polypeptide
containing at least one ornithine residue).
IV. In Vitro Method of Producing Ornithine-Containing
Polypeptide
[0140] Disclosed herein is an in vitro method of producing a
polypeptide with altered activity that includes at least one
ornithine residue in place of an arginine residue. Polypeptides
produced by such methods can be used in the therapeutic methods
provided herein to treat pulmonary disease. The method includes
contacting a polypeptide of interest including at least one
arginine residue susceptible to ADP-ribosylation with NAD and an
arginine-specific mono-ADP-ribosyltransferase (for example, ART1)
to produce a polypeptide including at least one ADP-ribosylated
arginine. The ADP-ribosylated polypeptide is incubated under
conditions sufficient for conversion of the at least one
ADP-ribosylated arginine to ornithine, and the ornithine-containing
polypeptide is isolated. In some examples, the polypeptide includes
an enzyme, a cytokine, a chemokine, an anti-inflammatory peptide, a
receptor ligand, a hormone, an antigen, or an antibody. In
particular examples, the polypeptide includes a defensin.
[0141] An ADP-ribose is covalently attached to another compound by
ADP-ribosylation. In one embodiment, the ART substrate (the source
of the ADP-ribose) is NAD. ADP-ribose acceptors of known mammalian
ARTs include basic and arginine-rich proteins that are involved in
host defense (e.g., lymphocyte activation, neutrophil chemotaxis).
ADP-ribose acceptors that contain an arginine can also be modified
by an ART, but need not be arginine rich or basic. ADP-ribose
acceptors include proteins with arginine residues, and thus
include, but are not limited to, alpha defensins (for example,
HNP-1, HNP-2, HNP-3, HNP-4, HD-5, HD-6, Def-X), beta defensins (for
example, hBD1, hBD-2, hBD-3, hBD-4), major basic protein,
eosinophil cationic protein, cathelicidin antimicrobial peptide
(e.g., hCAP18), and lysozyme. Thus, an ADP-ribosyltransferase (such
as ART1) can be used to produce ADP-ribosylated polypeptides, such
as defensin polypeptides.
[0142] In one example, the polypeptide, such as a defensin, which
includes at least one arginine residue (such as one, two, three, or
four arginine residues) that are susceptible to ADP-ribosylation is
contacted with NAD and an ART. In a particular example, the
polypeptide is a defensin such as HNP-1 (for example, SEQ ID NO:
2). ART (such as ART1) catalyzes transfer of an ADP-ribose from NAD
to an arginine residue in the polypeptide, such as HNP-1.
[0143] In some examples, the polypeptide, such as a defensin, is
ADP-ribosylated on at least one, at least two, at least three, or
at least four arginine residues. In a particular example, HNP-1 is
ADP-ribosylated on an arginine at position 14 of SEQ ID NO: 2. In
further examples, HNP-1 is ADP-ribosylated on an arginine at
position 24 of SEQ ID NO: 2. In additional examples, HNP-1 is
ADP-ribosylated at an arginine at position 14 and an arginine at
position 24 of SEQ ID NO: 2.
[0144] The disclosed methods include contacting a polypeptide such
as a defensin with at least one arginine residue susceptible to
ADP-ribosylation (for example, HNP-1) with NAD and an
arginine-specific mono-ADP-ribosyltransferase (for example, ART1)
to produce a polypeptide including at least one ADP-ribosylated
arginine. In some examples, purified polypeptide, such as a
defensin (such as recombinantly produced defensin polypeptide or
synthetic defensin polypeptide) is utilized. In one example,
synthetic HNP-1 is utilized (see e.g., Raj et al., Biochem. J.
347:633-641, 2000). Synthetic defensins are commercially available
(for example, Bachem, Torrance, Calif.; Catalog Nos. H-9855
(hHNP-1); H-9005 (hHNP-2); and H-9860 (hHNP-3)). In some examples,
about 1 nmol to 1 .mu.mol (for example, about 3 nmol, 5 nmol, 10
nmol, 50 nmol, 100 nmol, 250 nmol, 500 nmol, or 1 .mu.mol) of
polypeptide, such as a defensin polypeptide, is contacted with NAD
and ART to produce ADP-ribosylated polypeptide. In particular
examples, about 1 nmol to 25 nmol HNP-1 polypeptide (for example,
about 3 nmol, 5 nmol, 10 nmol, 15 nmol, or 20 nmol) is
ADP-ribosylated.
[0145] NAD is the source of ADP-ribose for ADP-ribosylation of a
polypeptide, such as a defensin polypeptide by ARTs. NAD is
commercially available (for example, Sigma-Aldrich, St. Louis, Mo.;
J T Baker, Phillipsburg, N.J.; Amresco, Solon, Ohio). In some
examples, about 1 mM to about 25 mM NAD (for example, about 2 mM,
about 5 mM, about 10 mM, about 15 mM, or about 20 mM) is incubated
with the polypeptide and ART for ADP-ribosylation of the
polypeptide.
[0146] ART catalyzes the transfer of ADP-ribose from NAD to a
polypeptide, such as a defensin polypeptide. Mammalian ART (for
example, ART1, ART2, ART3, ART4, or ART5) may be recombinantly
expressed in bacteria or mammalian cells and purified for use in
the in vitro methods described herein. In some examples, mammalian
ART1, such as human ART1 (for example, GenBank Accession No.
NP.sub.--004305, incorporated herein by reference as present in
GenBank on Sep. 10, 2009) or mouse ART1 (for example, GenBank
Accession No. NP.sub.--033840, incorporated herein by reference as
present in GenBank on Sep. 10, 2009) is used.
[0147] In some examples, mammalian ART1 can be introduced into a
bacterial expression vector and produced in E. coli. See, e.g.,
Paone et al., Proc. Natl. Acad. Sci. USA 99:8231-8235, 2002. In
other examples, mammalian ART1 may be introduced into a mammalian
expression vector and produced in a mammalian cell line. See, e.g.,
Paone et al., J. Biol. Chem. 281:17051-17060, 2006. In a
particular, non-limiting example, rat mammary adenocarcinoma (NMU)
cells are transfected with a plasmid containing mouse ART1. ART1 is
collected following treatment of intact cells with
phosphatidylinositol-specific phospholipase C (PI-PLC; for example,
0.05 U PI-PLC for 1 hour). Cells are sedimented, and the
supernatant containing released ART1 is collected.
[0148] ART activity can be assessed by monitoring transfer of
ADP-ribose to agmatine or by measuring the formation of
nicotinamide from NAD (see, e.g., Weng et al., J. Biol. Chem.
274:31797-31803, 1999; Okazaki et al., Blood. 88:915-21, 1996). In
some examples, about 1 nmol/h to about 20 nmol/h ART (for example,
about 1.5 nmol/h, about 2 nmol/h, about 5 nmol/h, about 10 nmol/h,
about 12 nmol/h or about 15 nmol/h) is incubated with a polypeptide
(such as a defensin polypeptide) and NAD for ADP-ribosylation of
the polypeptide.
[0149] Although exemplary amounts of polypeptide (such as HNP-1),
NAD, and ART are provided that can be used for in vitro
ADP-ribosylation of a polypeptide, one of skill in the art may
modify the amounts in the ADP-ribosylation reaction. For example,
the reaction may be scaled up using conventional techniques known
to one of skill in the art in order to increase the amount of
ADP-ribosylated defensin that is produced.
[0150] In the disclosed methods, the polypeptide, such as the
defensin, is contacted in vitro with NAD and ART to produce
ADP-ribosylated polypeptide. Conditions for ADP-ribosylation of a
polypeptide in vitro are known to one of skill in the art. In
particular examples, the reaction is carried out at a pH and
temperature suitable for ART activity. In some examples, the
reaction is carried out at approximately physiological pH, such as
about pH 7 to 8 (for example, about pH 7.0, 7.1, 7.2, 7.3, 7.4,
7.5, 7.6, 7.7., 7.8, 7.9, or 8.0) and at about 30.degree. C. to
about 37.degree. C. (for example, about 30.degree. C., 31.degree.
C., 32.degree. C., 33.degree. C., 34.degree. C., 35.degree. C.,
36.degree. C., or 37.degree. C.). The polypeptide, NAD, and ART are
incubated for a sufficient period of time for ADP-ribose to be
transferred to the polypeptide. In some examples, the incubation is
for about 2 hours to 24 hours (for example, about 4 hours, 8 hours,
12 hours, 16 hours, 18 hours, or 24 hours), such as overnight.
[0151] In a specific, non-limiting example, about 10 nmol HNP-1
polypeptide is mixed with about 5 mM NAD and about 12 nmol//h ART1
in potassium phosphate buffer of about pH 7.5, and the mixture is
incubated overnight at about 30.degree. C.
[0152] The ADP-ribosylated polypeptide (such as ADP-ribosylated
defensin, for example HNP-1) is incubated under conditions
sufficient for conversion of at least one ADP-ribosylated arginine
residue to an ornithine residue. As disclosed herein, the
conversion of ADP-ribosylated arginine HNP-1 to ornithine HNP-1 may
be a non-enzymatic process. The conditions sufficient for
conversion of ADP-ribosylated arginine to ornithine in vitro
include appropriate pH, time, and temperature, as described
herein.
[0153] In a particular example, an ADP-ribosylated arginine at
position 14 of HNP-1 (for example, position 14 of SEQ ID NO: 2) is
converted to an ornithine residue. In further examples, an
ADP-ribosylated arginine at position 24 of HNP-1 (for example,
position 24 of SEQ ID NO: 2) is converted to an ornithine residue.
In additional examples, ADP-ribosylated arginine residues at
position 14 and position 24 of HNP-1 (for example, positions 14 and
24 of SEQ ID NO: 2) are both converted to an ornithine residue.
However, any ADP-ribosylated arginine residue in a polypeptide
(such as an alpha defensin polypeptide) can be converted to an
ornithine residue by the methods described herein.
[0154] In some examples, the conditions sufficient for conversion
of at least one ADP-ribosylated arginine residue to an ornithine
residue include incubating the ADP-ribosylated protein (such as
ADP-ribosylated HNP-1) in a solution having a pH of about 7 to 9
(such as about pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9,
8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0). In
particular examples, the ADP-ribosylated protein is incubated in a
solution having a pH of about 7.5, about 8.0, or about 9.0. One of
skill in the art can select an appropriate solution for conversion
of ADP-ribosylated arginine to ornithine (for example, phosphate,
tris, acetate, citrate, or other buffers). In a particular example,
the solution includes potassium phosphate, such as about 1 mM to
about 500 mM (for example, about 10 mM to 100 mM) potassium
phosphate, for example about 50 mM potassium phosphate.
[0155] In some examples, the conditions for conversion of
ADP-ribosylated arginine to ornithine include incubating the
ADP-ribosylated protein (such as a defensin, for example HNP-1) for
a sufficient period of time for at least one ADP-ribosylated
arginine to be converted to an ornithine. In particular examples,
the incubation time is about 2 hours to about 168 hours (for
example, about 4 hours, about 8 hours, about 12 hours, about 16
hours, about 18 hours, about 24 hours, about 36 hours, about 48
hours, about 72 hours, about 96 hours, about 120 hours, about 144
hours, or about 168 hours). In one example, ADP-ribosylated
protein, such as HNP-1 (for example mono-ADP-ribosylated HNP-1 or
di-ADP-ribosylated HNP-1) is incubated overnight, for example,
about 16 to 18 hours, such that at least one ADP-ribosylated
arginine is converted to an ornithine. In another example,
ADP-ribosylated HNP-1 (such as mono-ADP-ribosylated-HNP-1 or
di-ADP-ribosylated-HNP-1) is incubated for about 24 hours, such
that at least one ADP-ribosylated arginine is converted to an
ornithine.
[0156] In some examples, the conditions for conversion of
ADP-ribosylated arginine to ornithine include incubating the
ADP-ribosylated protein (such as ADP-ribosylated HNP-1) at a
temperature sufficient for at least one ADP-ribosylated arginine to
be converted to an ornithine. In some examples, the incubation
temperature is about 30.degree. C. to about 37.degree. C. (for
example, about 30.degree. C., 31.degree. C., 32.degree. C.,
33.degree. C., 34.degree. C., 35.degree. C., 36.degree. C., or
37.degree. C.).
[0157] In one particular, non-limiting example, ADP-ribosylated
defensin (such as mono-ADP-ribosylated HNP-1 or di-ADP-ribosylated
HNP-1) is incubated in 50 mM potassium phosphate, pH 9 at
30.degree. C. overnight. In another example, ADP-ribosylated
defensin (such as mono-ADP-ribosylated HNP-1 or di-ADP-ribosylated
HNP-1) is incubated in 50 mM potassium phosphate, pH 7.5 at
30.degree. C. overnight.
[0158] In some examples, ADP-ribosylated polypeptide, such as a
defensin (for example mono-ADP-ribosylated HNP-1 or
di-ADP-ribosylated HNP-1) is purified prior to incubation under
conditions sufficient for conversion of at least one
ADP-ribosylated arginine residue to an ornithine residue. Methods
of purifying ADP-ribosylated proteins (such as defensins) are well
known to one of skill in the art. In some examples, ADP-ribosylated
protein (for example, mono-ADP-ribosylated HNP-1 and/or
di-ADP-ribosylated HNP-1) can be purified using chromatographic
methods (for example, HPLC or affinity chromatography). In other
examples, the polypeptide such as the defensin can be a recombinant
protein that includes a component that facilitates protein
purification, such as glutathione S-transferase, a polyhistidine
tag, or a FLAG or Myc tag. In a particular example, the
ADP-ribosylated protein is purified by HPLC.
[0159] In other examples, the conditions for ADP-ribosylation of at
least one arginine residue of a polypeptide by an ART in the
presence of NAD are the same as those sufficient for conversion of
at least one ADP-ribosylated arginine residue in the polypeptide to
an ornithine residue. In this case, ADP-ribosylated polypeptide,
such as the defensin is not purified prior to conversion of
ADP-ribosylated arginine to ornithine, rather, both reactions occur
in a single incubation. In a particular, non-limiting example, 10
nmol HNP-1 is incubated with 5 mM NAD and 12 nmol/h ART1 in 50 mM
potassium phosphate, pH 7.5 overnight at 30.degree. C.
[0160] The ornithine-containing polypeptide, such as the defensin
(such as ornithine-containing HNP-1) produced as described herein
may be isolated or purified. Methods of purifying polypeptides,
such as a polypeptide including at least one ornithine residue, are
well known to one of skill in the art. In particular examples, the
ornithine-containing protein (such as HNP-1 with at least one
arginine residue replaced with an ornithine residue) is purified
using chromatographic methods, (for example, HPLC or affinity
chromatography). In other examples, the ornithine-containing
defensin polypeptide may be a recombinant protein that includes a
component that facilitates protein purification, such as
glutathione S-transferase, a polyhistidine tag, or a FLAG or Myc
tag. In a particular example, the ornithine-containing protein is
purified by HPLC.
[0161] In some examples, the method further includes measuring or
assessing an activity of the modified polypeptide. The modification
of an arginine residue (for example, ADP-ribosylation) in a
polypeptide (such as a defensin) can alter its activity (such as
antimicrobial activity) and/or modify an immune response (see,
e.g., Paone et al., Proc. Natl. Acad. Sci. USA 99:8231-8235, 2002;
U.S. Pat. No. 7,511,015). In some examples, the
ornithine-containing polypeptide (such as ornithine-containing
HNP-1, for example, HNP-1 including at least one ornithine at amino
acid position 14 and/or 24 of SEQ ID NO: 2) has reduced
antimicrobial activity. In several non-limiting examples, the
decrease in antimicrobial activity is at least about a 20%
decrease, at least about a 50% decrease, at least about a 75%
decrease, at least about an 80% decrease, at least about a 90%
decrease, at least about a 95% decrease, at least about a 98%
decrease, or at least about a 100% decrease, for example relative
to a non-ornithine containing HNP-1. The antimicrobial activity can
be antibacterial, antifungal, or antiviral activity. Antimicrobial
activity can be determined by any method known to one of skill in
the art. In one example, the antimicrobial activity is lysis of
bacteria, fungi, or viruses.
[0162] In another example, the altered antimicrobial activity is an
increase in cytokine production. The increase in cytokine
production can be an increase in cytokine secretion, expression,
and/or release. In several non-limiting examples, the increase in
cytokine production is at least about a 20% increase, at least
about a 50% increase, at least about a 75% increase, at least about
an 80% increase, at least about a 90% increase, at least about a
1.5-fold increase, at least about a 2-fold increase, at least about
a 3-fold increase, or at least about a 5-fold increase, for example
relative to a non-ornithine containing HNP-1. Cytokine production
can be measured by any method known to one of skill in the art. In
one specific, non-limiting example, cytokine release is measured by
enzyme-linked immunosorbent assay (ELISA). In a particular example,
the cytokine is interleukin-8 (IL-8).
[0163] In a further example, the altered antimicrobial activity is
an increase in the recruitment of inflammatory cells. Recruitment
of inflammatory cells can be determined by any method known to one
of skill in the art. In one specific, non-limiting example, the
inflammatory cells are neutrophils. In a further embodiment, the
altered antimicrobial activity is an increase in inflammatory cell
chemotaxis. In several non-limiting examples, the increase in
inflammatory cell chemotaxis is at least about a 20% increase, at
least about a 50% increase, at least about a 75% increase, at least
about an 80% increase, at least about a 90% increase, at least
about a 1.5-fold increase, at least about a 2-fold increase, at
least about a 3-fold increase, or at least about a 5-fold increase,
for example relative to a non-ornithine containing HNP-1. In one
specific, non-limiting example the inflammatory cells are T cells.
T cell chemotaxis can be measured by any means known to one of
skill in the art, for example, measuring the length of migration of
T cells, the number of migrating T cells, or both. In a specific
example, T cell migration is measured in vitro, such as by
measuring T cell migration from one cell culture chamber to another
cell culture chamber through a porous membrane.
[0164] In some examples, the antimicrobial activity of a modified
alpha defensin is altered as compared to an unmodified (native)
alpha defensin polypeptide or an ADP-ribosylated alpha defensin
polypeptide. In one specific, non-limiting example, the modified
alpha defensin is an ornithine-containing HNP-1 polypeptide and the
unmodified alpha defensin is unmodified HNP-1 polypeptide. In
another specific, non-limiting example, the modified alpha defensin
is an ornithine-containing HNP-1 polypeptide and the
ADP-ribosylated alpha defensin is ADP-ribosylated HNP-1
polypeptide.
V. Pharmaceutical Compositions and Administration
[0165] Pharmaceutical compositions that include a polypeptide with
at least one arginine residue susceptible to ADP-ribosylation, or a
modified polypeptide with at least one ornithine residue in place
of an arginine residue, and/or pharmaceutical compositions that
include NAD can be formulated with an appropriate solid or liquid
carrier, depending upon the particular mode of administration
chosen. A specific, non-limiting example of a polypeptide including
at least one arginine residue susceptible to ADP-ribosylation
includes HNP-1 polypeptide (for example, SEQ ID NO: 2). A specific,
non-limiting example of a modified polypeptide including at least
one ornithine residue in place of an arginine residue includes an
HNP-1 polypeptide including an ornithine residue at amino acid
position 14 and/or 24 of SEQ ID NO: 2. In one example, the
pharmaceutical composition includes at least one polypeptide (such
as a defensin, for example, HNP-1) and NAD. In additional examples,
the pharmaceutical composition further includes an ART (such as
ART1, ART2, or ART5).
[0166] The pharmaceutically acceptable carriers and excipients
useful in this disclosure are conventional. See, e.g., Remington:
The Science and Practice of Pharmacy, The University of the
Sciences in Philadelphia, Editor, Lippincott, Williams, &
Wilkins, Philadelphia, Pa., 21.sup.st Edition (2005). For instance,
parenteral formulations usually comprise injectable fluids that are
pharmaceutically and physiologically acceptable fluid vehicles such
as water, physiological saline, other balanced salt solutions,
aqueous dextrose, glycerol or the like. For solid compositions
(e.g., powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically-neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, pH buffering agents, or the like, for
example sodium acetate or sorbitan monolaurate. Excipients that can
be included are, for instance, other proteins, such as human serum
albumin or plasma preparations.
[0167] Medicinal and pharmaceutical agents, for instance
immunostimulants, also can be included. Immunostimulants include,
but are not limited to, cytokines, such as Macrophage Inflammatory
Protein (MIP)-, IL-1, IL-8, IL-10, granulocyte-macrophage colony
stimulating factor (GM-CSF), granulocyte colony stimulating factor
(G-CSF), neurokinin, and tumor necrosis factor-alpha (TNF.alpha.),
for example.
[0168] The dosage form of the pharmaceutical composition will be
determined by the mode of administration chosen. For instance, in
addition to injectable fluids, topical, inhalation, oral and
suppository formulations can be employed. Topical preparations can
include eye drops, ointments, sprays, patches and the like.
Inhalation preparations can be liquid (e.g., solutions or
suspensions) and include mists, sprays and the like. Oral
formulations can be liquid (e.g., syrups, solutions or
suspensions), or solid (e.g., powders, pills, tablets, or
capsules). Suppository preparations can also be solid, gel, or in a
suspension form. For solid compositions, conventional non-toxic
solid carriers can include pharmaceutical grades of mannitol,
lactose, starch, or magnesium stearate. Actual methods of preparing
such dosage forms are known, or will be apparent, to those skilled
in the art.
[0169] The pharmaceutical compositions that include a polypeptide
(such as a defensin, for example, HNP-1) including at least one
arginine residue susceptible to ADP-ribosylation or a modified
polypeptide including at least one ornithine residue in place of an
arginine residue (such as a modified defensin) can be formulated in
unit dosage form, suitable for individual administration of precise
dosages. In one specific, non-limiting example, a unit dosage can
contain from about 1 ng to about 1 mg of HNP-1 polypeptide. In
another non-limiting example, a unit dosage can contain from about
0.5 .mu.M to about 200 mM NAD. The amount of active compound(s)
administered will be dependent on the subject being treated, the
severity of the affliction, and the manner of administration, and
is best left to the judgment of the prescribing clinician. Within
these bounds, the formulation to be administered will contain a
quantity of the active component(s) in amounts effective to achieve
the desired effect in the subject being treated.
[0170] The compounds of this disclosure can be administered to
humans or other animals on whose tissues they are effective in
various manners such as topically, orally, intravenously,
intramuscularly, intraperitoneally, intranasally, intradermally,
intrathecally, subcutaneously, via inhalation or via suppository.
The particular mode of administration and the dosage regimen will
be selected by the attending clinician, taking into account the
particulars of the case (e.g. the subject, the disease, the disease
state involved, and whether the treatment is prophylactic).
Treatment can involve daily or multi-daily doses of compound(s)
over a period of a few days to months, or even years.
[0171] Site-specific administration of the disclosed compounds can
be used, for instance by administering a NAD and/or a polypeptide
to the lungs or respiratory tract to treat a subject with pulmonary
disease. By way of example, one method of administration to the
lungs of an individual is by inhalation through the use of a
nebulizer or inhaler. For example, the NAD and/or polypeptide is
formulated in an aerosol or particulate and drawn into the lungs
using a standard nebulizer well known to those skilled in the art.
Other routes of administration to the lungs or respiratory tract
include bronchial, intranasal, or other inhalatory routes. In some
examples the NAD and/or polypeptide is administered by inhalation
(for example, by inhaling an aerosol); direct installation in the
lung via a bronchoscope, endotracheal tube, or an artificial
ventilation device; nasal administration (intranasal or
transnasal); bronchial, or intratracheally (for example, by
injection directly into the trachea or tracheostomy).
[0172] A therapeutically effective amount of NAD administered by
inhalation in conjunction with administration of a polypeptide
(such as a defensin, for example HNP-1) having at least one
arginine residue susceptible to ADP-ribosylation can be the amount
of NAD necessary to treat a pulmonary disease of a subject, for
example, an amount necessary to increase ADP-ribosylation of a
polypeptide and its subsequent conversion to a polypeptide
containing at least one ornithine residue in place of an arginine
residue.
[0173] A therapeutically effective amount of a polypeptide
including at least one arginine residue susceptible to
ADP-ribosylation and NAD (administered with or without an ART
polypeptide) or a modified polypeptide including at least one
ornithine residue in place of an arginine residue, can be the
amount of a polypeptide (such as a defensin, for example, HNP-1)
including at least one arginine residue susceptible to
ADP-ribosylation and NAD or a modified polypeptide (such as a
defensin, for example, HNP-1) including at least one ornithine
residue in place of an arginine residue necessary to treat a
pulmonary disease of a subject.
[0174] A therapeutically effective amount of a polypeptide
including at least one arginine residue susceptible to
ADP-ribosylation and NAD (administered with or without an ART
polypeptide) or a modified polypeptide including at least one
ornithine residue in place of an arginine residue, can be
administered in a single dose, or in several doses, for example
daily, during a course of treatment. However, the therapeutically
effective amount will be dependent on the subject being treated,
the severity and type of the affliction, and the manner of
administration of the therapeutic(s).
[0175] The present disclosure also includes combinations of a
polypeptide including at least one arginine residue susceptible to
ADP-ribosylation and NAD (administered with or without ART) or a
modified polypeptide including at least one ornithine residue in
place of an arginine residue, with one or more other agents useful
in the treatment of pulmonary disease. For example, the compounds
of this disclosure can be administered in combination with
effective doses of modified antimicrobial agents other than
defensins, immunostimulants, anti-tumor agents, anti-inflammatory
agents, anti-infectives, and/or vaccines. The term "administration
in combination" or "co-administration" refers to both concurrent
and sequential administration of the active agents.
[0176] In one example, a polypeptide including at least one
arginine residue susceptible to ADP-ribosylation and NAD
(administered with or without ART) or a modified polypeptide
including at least one ornithine residue in place of an arginine
residue may be co-administered with therapeutic that is effective
to decrease the viscosity of pulmonary secretions, such as a DNase
(for example, DNase I or DNase II). See, e.g., U.S. Pat. No.
7,297,526. The DNase may be administered by any suitable route, for
example, by inhalation or direct instillation in the nasotracheal
or endotracheal tubes in an intubated patient. In a particular
example, a patient having a pulmonary disease with abnormal or
viscous secretions (including, but not limited to pneumonia,
chronic bronchitis, cystic fibrosis, or asthma) is treated with a
polypeptide (such as a defensin) including at least one residue
susceptible to ADP-ribosylation, NAD and DNase. In another example,
a patient having a pulmonary disease with abnormal or viscous
secretions (including, but not limited to pneumonia, chronic
bronchitis, cystic fibrosis, or asthma) is treated with a
polypeptide (such as a defensin) including at least one residue
susceptible to ADP-ribosylation, NAD, ART, and DNase. In a further
example, a patient having a pulmonary disease with abnormal or
viscous secretions (including, but not limited to pneumonia,
chronic bronchitis, cystic fibrosis, or asthma) is treated with a
polypeptide (such as a defensin) including at least one ornithine
residue in place of an arginine residue and DNase.
[0177] A subject that has a pulmonary disease (such as cystic
fibrosis, emphysema, asthma, sarcoidosis, chronic bronchitis,
bronchopulmonary dysplasia, pulmonary fibrosis, pneumonia, or adult
respiratory distress syndrome), will be a candidate for treatment
using the therapeutic methods disclosed herein.
[0178] The disclosure is illustrated by the following non-limiting
Examples.
EXAMPLES
Example 1
Conversion of ADP-Ribosylated Arginine to Ornithine in HNP-1
[0179] This example describes the non-enzymatic conversion of
ADP-ribosylated arginine in HNP-1 to ornithine.
Methods
[0180] ADP-ribosylation of HNP-1:
[0181] Rat mammary adenocarcinoma (NMU) cells transfected with
plasmids containing mART1 were grown in Eagle's minimal essential
medium with 10% fetal bovine serum (Invitrogen) and 0.5 mg/ml
Geneticin (G-418). Cells were purchased from American Type Culture
Collection (Manassas, Va.). Protein released from the cells by
phosphatidyl inositol-specific phospholipase C, collected in the
medium for ADP-ribosyltransferase activity was assayed by
quantifying the transfer of ADP-ribose to agmatine in standard
assays as described (Paone et al., J. Biol. Chem., 281:17054-17060,
2006).
[0182] HNP-1 was incubated overnight at 30.degree. C. with 5 mM NAD
in 150 .mu.l of 50 mM potassium phosphate, pH 7.5 and ART1.
Reactions were terminated by addition of guanidine HCl to a final
concentration of 6 M. Products were separated by HPLC and analyzed
by mass spectrometry (MS).
[0183] Preparation of ADP-ribosyl-[.sup.14C]arginine:
[0184] Cholera toxin A subunit (60 .mu.g), 30 mM dithiothreitol
(DTT), 10 mM NAD, and 10 mM arginine (0.5 .mu.Ci .sup.14C/assay)
were incubated overnight at 30.degree. C. with 30 .mu.g ovalbumin
in 20 mM potassium phosphate, pH 7.5, (volume 300 .mu.l). Reaction
products were separated on a strong anion exchange (SAX) column
(DuPont, Wilmington, Del.) by gradient elution (Paone et al., J.
Biol. Chem., 281:17054-17060, 2006). Radioactive peaks were
collected, vacuum-concentrated, and applied to a Discovery BioWide
Pore C18 RP-HPLC column (Supelco, Bellefonte, Pa.) equilibrated for
15 min with HPLC water, 0.05% TFA, (flow=0.8 ml/min), followed by a
5 min linear gradient of 0 to 100% acetonitrile. Peaks, monitored
by absorbance at 254 nm and radioactivity identified as
ADP-ribosyl-[.sup.14C]arginine were confirmed by MS analysis.
Samples (25 .mu.l) of ADP-ribosyl-[.sup.14C]arginine were
vacuum-dried, dissolved in 200 .mu.l 6N HCl (Fluka), and hydrolyzed
under nitrogen (155.degree. C., 45 min) to release arginine. The
hydrolysate was vacuum-dried, dissolved in 25 .mu.l 0.05% TFA and
subjected to o-phthalaldehyde (OPA) derivatization (Agilent
Technologies, Santa Clara, Calif.) before HPLC separation. The
arginine peak was quantified by absorbance at 338 nm and
fluorescence (340 nm excitation/450 nm emission) then compared to a
standard curve. Similarly, the amount of ornithine following
incubation of ADP-ribosylarginine samples was determined by OPA
derivatization and compared to a standard curve.
[0185] Amino Acid Analysis:
[0186] HNP-1 (Bachem, Torrance, Calif.) was vacuum-dried, dissolved
in 200 .mu.l of 6N HCl plus 5 .mu.l of 40 mM DTT before hydrolysis
under nitrogen at 155.degree. C. for 45 min. The hydrolysate was
vacuum-dried and solubilized in 25 .mu.l water, 0.05% TFA before
OPA pre-column automated derivatization. The conditions for the
derivatization reaction and the HPLC separation (with the
modification below) are described in Agilent Technologies Technical
Note (Publication number 5980-1193EN). The HPLC column, Eclipse-AAA
(4.6.times.150 mm, 5 .mu.m particle size) was equilibrated with
mobile phase A, 40 mM sodium dibasic phosphate buffer pH 7.8 using
a linear gradient of 0 to 40% of phase B, acetonitrile:MeOH:water
(45:45:10) for 1.9-15 min; 15-18.1 min gradient to 57% B, 18.1-18.6
min gradient to 100% B.
[0187] MS and Sequence Analysis:
[0188] HNP-1 was reduced, cleaved by trypsin, and analyzed by
reverse phase chromatography-mass spectrometry as described (Paone
et al., Proc. Natl. Acad. Sci. USA 99:8231-8235, 2002), except that
the reverse phase column was a Zorbax 300SB-C18, 2.1.times.50 mm,
3.5 micron, and the mass spectrometer was an Agilent model G1969
with a time of flight detector. Mass spectra were deconvoluted with
Agilent software MassHunter version 2, and the fraction of each
species was calculated from the areas of the deconvoluted
peaks.
Results
[0189] HNP-1 was ADP-ribosylated in vitro using purified ART1. HPLC
analysis of the reaction products identified HNP-1,
mono-ADP-ribosylated HNP-1, and di-ADP-ribosylated HNP-1 (FIG. 1).
Incubation of ART1 (2 nmol/h activity) and HNP-1 for 24 hours at
30.degree. C. decreased the amount of di-ADP-ribosylated HNP-1 and
increased the amount of a fourth HPLC peak (FIG. 1). MS analysis
identified HNP-1, mono-ADP-HNP-1 ribosylated on arginine 14, and
di-ribosylated HNP-1 with a second modification of arginine 24. The
purified 3940 Da product, identified as
ADP-ribosylated-HNP-1-ornithine, mapped to position 14 was
subjected to acid hydrolysis. Amino acid analysis confirmed the
presence of ornithine in the modified, but not in the substrate
HNP-1 (FIG. 2). Since arginine 14 was the site of the initial
ADP-ribosylation, it appeared that ADP-ribosyl-arginine was the
precursor of ornithine in HNP-1. Amounts of modified
HNP-1-ornithine recovered in the reaction mix increased with
incubation time, reaching about 26% after 168 hours, consistent
with non-enzymatic conversion of arginine to ornithine (FIG.
3).
[0190] To verify that ART1 (or an enzymatic contaminant) was not
required for ornithine formation, HPLC-purified mono- and
di-modified HNP-1 were incubated for 24 hours at 37.degree. C. at
pH 7 or 9 and the reaction products were analyzed by HPLC (FIG. 4,
Table 1). Di-ADP-ribosylated-HNP-1 degraded to
mono-ADP-ribosylated-HNP-1-ornithine of 3940 molecular mass at pH 7
and the amount was greater at pH 9 (FIG. 4A-D). About 50% of
di-ADP-ribosylated-HNP-1 degraded to
mono-ADP-ribosylated-HNP-1-ornithine at pH 9, with ADP-ribosylated
arginine 14 and ornithine at position 24 or ADP-ribosylated
arginine 24 and ornithine at position 14. Purified
mono-ADP-ribosylated-HNP-1 was converted to HNP-1-ornithine (3399.5
Da) under the same conditions at pH 7 and the amount was greater at
pH 9 (FIG. 4E-G).
TABLE-US-00014 TABLE 1 MS analysis of mono- and di-ADP-ribosyl-
HNP-1 incubated at pH 7 or 9 Di- ADPR- Di- Time ADPR- HNP- Orn-
HNP- ADPR- pH (h) HNP HNP Orn HNP Orn HNP ADPR- 7 0 0 100 0 0 0 0
HNP ADPR- 7 24 0 82 .+-. 13 18 .+-. 13 0 0 0 HNP ADPR- 9 0 0 100 0
0 0 0 HNP ADPR- 9 24 0 61 .+-. 9 39 .+-. 9 0 0 0 HNP Di- 7 0 0 0 0
0 100 ADPR- HNP Di- 7 24 0 0 0 1 .+-. 1 32 .+-. 17 67 .+-. 18 ADPR-
HNP Di- 9 0 0 0 0 0 13 87 ADPR- HNP Di- 9 24 0 0 0 9 .+-. 3 55 .+-.
3 36 .+-. 0 ADPR- HNP Values indicate the percent of each species
at the end of the incubation
[0191] Arginase, found predominantly in liver, catalyzes the
hydrolysis of arginine to ornithine in the urea cycle. The enzyme
has been reported to require free arginine (Jenkinson et al., Comp.
Biochem. Physiol. B Biochem. Mol. Biol. 114:107-132, 1996). HNP-1
was tested as a potential substrate for arginase. HNP-1 (1.4 nmol),
mono-ADP-ribosylated-HNP-1 (1 nmol), ADP-ribosylarginine (18
.mu.M), and arginine (5 mM) were each incubated with
manganese-activated arginase (0.7 U) at pH 9.5 in 200 .mu.l for 10
minutes at 37.degree. C. Identification of the products by amino
acid analysis and MS identified ornithine only in the reaction that
contained free arginine. This confirms that HNP-1 is not a
substrate for arginase.
[0192] Ornithine has been found in acid hydrolysates of human skin
collagen and lens crystalline in amounts that increase with the age
of subjects from whom the proteins were isolated (Sell and Monnier,
J. Biol. Chem. 279:54173-54184, 2004). The presence of ornithine in
these proteins is believed to result from the reaction of reducing
sugars with arginine residues to form advanced glycation end
products (AGEs), followed by time-dependent breakdown of the AGEs
to yield ornithine. Non-enzymatic modification of several proteins
by sugars in vitro has been reported (Shapiro et al., J. Biol.
Chem. 255:3120-3127, 1980; Sun et al., J. Agric. Food Chem.
52:1293-1299, 2004), but ornithine in a primary protein sequence
has only been reported only in collagen as a result of age-related
glycation. No ornithine was identified after incubation of arginine
with free ADP-ribose (FIG. 5A); ornithine alone is shown in FIG.
5B. Arginine was cleaved from ADP-ribosylarginine incubated in 6N
HCl at 37.degree. C. for 24 hours (FIG. 5C). However, consistent
with non-enzymatic conversion of modified arginine to ornithine,
ADP-ribosylarginine generated ornithine when incubated at
37.degree. C. at pH 9 for 24 hours (compare FIGS. 5D and 5E). These
data demonstrated that the amino acid sequence of HNP-1 is not
required for conversion of modified arginines to ornithine. In
contrast to the enzymatic cleavage of ADP-ribosylarginine by
ADP-ribosyl-arginine hydrolase-1 (ARH1) at carbon 1'' of
ADP-ribose, which releases ADP-ribose from arginine, the
non-enzymatic hydrolysis of ADP-ribose-arginine at the guanidino
carbon of arginine produced ornithine.
[0193] Amino acid analysis of ADP-ribosyl-[.sup.14C]-arginine at
time zero showed the presence of ADP-ribosyl-[.sup.14C]-arginine
and arginine (FIG. 5F). Amino acid analysis of
ADP-ribosyl-[.sup.14C]-arginine incubated for 24 hours at
37.degree. C., pH 9 showed the accumulation of ornithine (FIGS. 5G
and 5H). The maximal amount of ornithine quantified by amino acid
analysis without prior acid hydrolysis was 21% of initial
ADP-ribosylarginine after incubation at pH 9 for 24 hours. After
incubation at pH 7 or pH 8, the amount of ornithine was less than
at pH 9 and was generated at a slower rate.
Example 2
Isolation of Ornithine-Containing HNP-1 from Bronchoalveolar
Lavage
[0194] This example describes the detection of ornithine containing
HNP-1 in BALF from a patient with IPF.
[0195] Preparation of bronchoalveolar lavage fluid (BALF) was as
previously described (Paone et al., J. Biol. Chem.,
281:17054-17060, 2006). The clinical protocol (protocol 99-H-0068)
was approved by the National Institutes of Health Institutional
Review Board. Written informed consent was obtained from all
subjects. BALF samples were obtained from seven patients with IPF
and four patients with asthma. Briefly, 8 ml of BALF was applied to
LC-18 SUPELCLEAN.TM. SPE tubes (Sigma-Aldrich, St. Louis, Mo.)
equilibrated in 10% isopropanol/0.1% TFA, washed, and eluted with
50% isopropanol/0.1% TFA. The eluted proteins were
vacuum-concentrated before separation by RP-HPLC as described in
Example 1.
[0196] Mono- and di-ADP-ribosylated-HNP-1 have previously been
detected in BALF from patients with IPF and asthma (Paone et al.,
J. Biol. Chem. 281:17054-17060, 2006). In four of the seven IPF
samples, a broad peak eluted at the retention time of HNP-1. MS
analysis confirmed that one of the samples contained HNP-1 and
consisted of 38.8% HNP-1, 32.1% di-ADP-ribosylated HNP-1, 20.8%
ADP-ribosyl-HNP-1 and 8.3% ADP-ribosyl-HNP-1-orninthine. These data
are consistent with the in vivo alteration of HNP-1 primary
sequence. Of note, as with in vitro ADP-ribosyl-HNP-1, in the in
vivo modified material, di-ADP-ribosylated HNP-1-ornithine was not
detected, consistent with ornithine is not ADP-ribosylated by
NAD:arginine ADP-ribosyltransferases. Experimentally arginine and
agmatine served as substrates for ART1, but ornithine did not.
HNP-1-ornithine was not detected in BALF from patients with asthma
(n=4); of the four patients, one had both di- and
mono-ADP-ribosylated HNP-1.
Example 3
Treatment of Pulmonary Disease with Defensin and NAD
[0197] This example describes exemplary methods for treating a
subject with pulmonary disease with a defensin polypeptide and NAD.
However, one skilled in the art will appreciate that methods that
deviate from these specific methods can also be used to
successfully treat pulmonary disease.
[0198] Based upon the teaching disclosed herein, pulmonary disease
(for example, cystic fibrosis, emphysema, asthma, sarcoidosis,
chronic bronchitis, bronchopulmonary dysplasia, pulmonary fibrosis,
pneumonia, or adult respiratory distress syndrome), can be treated
by administering a therapeutically effective amount of a defensin
polypeptide including at least one arginine residue susceptible to
ADP-ribosylation and NAD, wherein the NAD is administered by
inhalation.
[0199] In one example, a clinical trial includes half of the
subjects following an established protocol for treatment of
pulmonary disease or alternatively, a placebo control. The other
half is treated by administering a defensin polypeptide including
at least one arginine residue susceptible to ADP-ribosylation and
NAD, wherein the NAD is administered by inhalation.
[0200] A therapeutically effective dose of the defensin polypeptide
including at least one arginine residue susceptible to
ADP-ribosylation and a therapeutically effective dose of NAD is
administered to the subject (such as a subject either at risk for
developing a pulmonary disease or known to have a pulmonary
disease). Additional agents, such as immune stimulatory agents, can
also be administered to the subject simultaneously, prior to, or
following administration of the disclosed agents. The NAD is
administered by inhalation. Administration of the defensin
polypeptide can be achieved by any method known in the art, such as
oral, inhalation, intravenous, intramuscular, intraperitoneal, or
subcutaneous administration.
[0201] The amount of the defensin polypeptide and NAD administered
to treat the pulmonary disease depends on the subject being
treated, the severity of the disorder, and the manner of
administration of the therapeutic composition. Ideally, a
therapeutically effective amount of an agent is the amount
sufficient to prevent, reduce, and/or inhibit, and/or treat the
condition (e.g., the pulmonary disease) in a subject without
causing a substantial cytotoxic effect in the subject. An effective
amount can be readily determined by one skilled in the art, for
example using routine trials establishing dose response curves. In
addition, particular exemplary dosages are provided above. The
therapeutic compositions can be administered in a single dose
delivery, via continuous delivery over an extended time period, in
a repeated administration protocol (for example, by a daily,
weekly, or monthly repeated administration protocol). In one
example, a therapeutic agent that includes a defensin polypeptide
and NAD is administered via inhalation to a subject. As such, these
compositions may be formulated with an inert diluent or with a
pharmaceutically acceptable carrier. Administration of the
therapeutic compositions can be taken long term (for example over a
period of months or years).
[0202] Following the administration of one or more therapies,
subjects having pulmonary disease can be monitored for reductions
in one or more clinical symptoms associated with the pulmonary
disease. In particular examples, subjects are analyzed one or more
times, starting 7 days following treatment. Subjects can be
monitored using any method known in the art. For example, lung
function of the subject can be assessed by measuring forced vital
capacity (FVC), forced expiratory volume (FEV), or forced
expiratory flow (FEF). Other signs and symptoms of the particular
pulmonary disease can also be assessed. A reduction in the clinical
symptoms associated with the pulmonary disease, for example,
improved lung function indicates the effectiveness of the
treatment.
[0203] One of skill in the art will appreciate that the disclosed
agents including defensin polypeptides (or modified defensin
polypeptides including at least one ornithine) can be tested for
safety in animals, and then used for clinical trials in animals or
humans. In one example, animal models of pulmonary disease are
employed to determine therapeutic value of the disclosed
agents.
[0204] In view of the many possible embodiments to which the
principles of the disclosure may be applied, it should be
recognized that the illustrated embodiments are only examples and
should not be taken as limiting the scope of the invention. Rather,
the scope of the invention is defined by the following claims. We
therefore claim as our invention all that comes within the scope
and spirit of these claims.
Sequence CWU 1
1
13194PRTHomo sapiens 1Met Arg Thr Leu Ala Ile Leu Ala Ala Ile Leu
Leu Val Ala Leu Gln 1 5 10 15 Ala Gln Ala Glu Pro Leu Gln Ala Arg
Ala Asp Glu Val Ala Ala Ala 20 25 30 Pro Glu Gln Ile Ala Ala Asp
Ile Pro Glu Val Val Val Ser Leu Ala 35 40 45 Trp Asp Glu Ser Leu
Ala Pro Lys His Pro Gly Ser Arg Lys Asn Met 50 55 60 Ala Cys Tyr
Cys Arg Ile Pro Ala Cys Ile Ala Gly Glu Arg Arg Tyr 65 70 75 80 Gly
Thr Cys Ile Tyr Gln Gly Arg Leu Trp Ala Phe Cys Cys 85 90
230PRTHomo sapiens 2Ala Cys Tyr Cys Arg Ile Pro Ala Cys Ile Ala Gly
Glu Arg Arg Tyr 1 5 10 15 Gly Thr Cys Ile Tyr Gln Gly Arg Leu Trp
Ala Phe Cys Cys 20 25 30 329PRTHomo sapiens 3Cys Tyr Cys Arg Ile
Pro Ala Cys Ile Ala Gly Glu Arg Arg Tyr Gly 1 5 10 15 Thr Cys Ile
Tyr Gln Gly Arg Leu Trp Ala Phe Cys Cys 20 25 494PRTHomo sapiens
4Met Arg Thr Leu Ala Ile Leu Ala Ala Ile Leu Leu Val Ala Leu Gln 1
5 10 15 Ala Gln Ala Glu Pro Leu Gln Ala Arg Ala Asp Glu Val Ala Ala
Ala 20 25 30 Pro Glu Gln Ile Ala Ala Asp Ile Pro Glu Val Val Val
Ser Leu Ala 35 40 45 Trp Asp Glu Ser Leu Ala Pro Lys His Pro Gly
Ser Arg Lys Asn Met 50 55 60 Asp Cys Tyr Cys Arg Ile Pro Ala Cys
Ile Ala Gly Glu Arg Arg Tyr 65 70 75 80 Gly Thr Cys Ile Tyr Gln Gly
Arg Leu Trp Ala Phe Cys Cys 85 90 530PRTHomo sapiens 5Asp Cys Tyr
Cys Arg Ile Pro Ala Cys Ile Ala Gly Glu Arg Arg Tyr 1 5 10 15 Gly
Thr Cys Ile Tyr Gln Gly Arg Leu Trp Ala Phe Cys Cys 20 25 30
697PRTHomo sapiens 6Met Arg Ile Ile Ala Leu Leu Ala Ala Ile Leu Leu
Val Ala Leu Gln 1 5 10 15 Val Arg Ala Gly Pro Leu Gln Ala Arg Gly
Asp Glu Ala Pro Gly Gln 20 25 30 Glu Gln Arg Gly Pro Glu Asp Gln
Asp Ile Ser Ile Ser Phe Ala Trp 35 40 45 Asp Lys Ser Ser Ala Leu
Gln Val Ser Gly Ser Thr Arg Gly Met Val 50 55 60 Cys Ser Cys Arg
Leu Val Phe Cys Arg Arg Thr Glu Leu Arg Val Gly 65 70 75 80 Asn Cys
Leu Ile Gly Gly Val Ser Phe Thr Tyr Cys Cys Thr Arg Val 85 90 95
Asp 734PRTHomo sapiens 7Val Cys Ser Cys Arg Leu Val Phe Cys Arg Arg
Thr Glu Leu Arg Val 1 5 10 15 Gly Asn Cys Leu Ile Gly Gly Val Ser
Phe Thr Tyr Cys Cys Thr Arg 20 25 30 Val Asp 894PRTHomo sapiens
8Met Arg Thr Ile Ala Ile Leu Ala Ala Ile Leu Leu Val Ala Leu Gln 1
5 10 15 Ala Gln Ala Glu Ser Leu Gln Glu Arg Ala Asp Glu Ala Thr Thr
Gln 20 25 30 Lys Gln Ser Gly Glu Asp Asn Gln Asp Leu Ala Ile Ser
Phe Ala Gly 35 40 45 Asn Gly Leu Ser Ala Leu Arg Thr Ser Gly Ser
Gln Ala Arg Ala Thr 50 55 60 Cys Tyr Cys Arg Thr Gly Arg Cys Ala
Thr Arg Glu Ser Leu Ser Gly 65 70 75 80 Val Cys Glu Ile Ser Gly Arg
Leu Tyr Arg Leu Cys Cys Arg 85 90 932PRTHomo sapiens 9Ala Thr Cys
Tyr Cys Arg Thr Gly Arg Cys Ala Thr Arg Glu Ser Leu 1 5 10 15 Ser
Gly Val Cys Glu Ile Ser Gly Arg Leu Tyr Arg Leu Cys Cys Arg 20 25
30 10100PRTHomo sapiens 10Met Arg Thr Leu Thr Ile Leu Thr Ala Val
Leu Leu Val Ala Leu Gln 1 5 10 15 Ala Lys Ala Glu Pro Leu Gln Ala
Glu Asp Asp Pro Leu Gln Ala Lys 20 25 30 Ala Tyr Glu Ala Asp Ala
Gln Glu Gln Arg Gly Ala Asn Asp Gln Asp 35 40 45 Phe Ala Val Ser
Phe Ala Glu Asp Ala Ser Ser Ser Leu Arg Ala Leu 50 55 60 Gly Ser
Thr Arg Ala Phe Thr Cys His Cys Arg Arg Ser Cys Tyr Ser 65 70 75 80
Thr Glu Tyr Ser Tyr Gly Thr Cys Thr Val Met Gly Ile Asn His Arg 85
90 95 Phe Cys Cys Leu 100 1130PRTHomo sapiens 11Thr Cys His Cys Arg
Arg Ser Cys Tyr Ser Thr Glu Tyr Ser Tyr Gly 1 5 10 15 Thr Cys Thr
Val Met Gly Ile Asn His Arg Phe Cys Cys Leu 20 25 30 1294PRTHomo
sapiens 12Met Arg Thr Leu Thr Leu Leu Ser Ala Phe Leu Leu Val Ala
Leu Gln 1 5 10 15 Ala Trp Ala Glu Pro Leu Gln Ala Arg Ala His Glu
Met Pro Ala Gln 20 25 30 Lys Gln Pro Pro Ala Asp Asp Gln Asp Val
Val Ile Tyr Phe Ser Gly 35 40 45 Asp Asp Ser Cys Ser Leu Gln Val
Pro Gly Ser Thr Lys Gly Leu Ile 50 55 60 Cys His Cys Arg Val Leu
Tyr Cys Ile Phe Gly Glu His Leu Gly Gly 65 70 75 80 Thr Cys Phe Ile
Leu Gly Glu Arg Tyr Pro Ile Cys Cys Tyr 85 90 1331PRTHomo sapiens
13Ile Cys His Cys Arg Val Leu Tyr Cys Ile Phe Gly Glu His Leu Gly 1
5 10 15 Gly Thr Cys Phe Ile Leu Gly Glu Arg Tyr Pro Ile Cys Cys Tyr
20 25 30
* * * * *