U.S. patent application number 16/692849 was filed with the patent office on 2020-03-12 for lipoprotein targeting protease inhibitors and uses.
This patent application is currently assigned to The United States of America, as represented by the Secretary, Dept. of Health and Human Services. The applicant listed for this patent is The United States of America, as represented by the Secretary, Dept. of Health and Human Services, The United States of America, as represented by the Secretary, Dept. of Health and Human Services. Invention is credited to Scott M. Gordon, Alan T. Remaley.
Application Number | 20200078434 16/692849 |
Document ID | / |
Family ID | 60242757 |
Filed Date | 2020-03-12 |
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United States Patent
Application |
20200078434 |
Kind Code |
A1 |
Remaley; Alan T. ; et
al. |
March 12, 2020 |
LIPOPROTEIN TARGETING PROTEASE INHIBITORS AND USES
Abstract
Described herein is the design and construction of a class of
lipoprotein targeting protease inhibitors. Small peptides with
protease inhibitor activity are conjugated to hydrophobic,
lipoprotein targeting molecules using, for instance, amine reactive
chemistry. Methods of use of the resultant lipoprotein targeting
protease inhibitor (antiprotease) molecules are also described.
Also described is the production and use of protease inhibitor
enriched HDL particles, as well as A1AT-peptide-enriched HDL
particles, and their use in various therapeutic contexts.
Inventors: |
Remaley; Alan T.; (Bethesda,
MD) ; Gordon; Scott M.; (Lexington, KY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Dept. of Health and Human Services |
Bethesda |
MD |
US |
|
|
Assignee: |
The United States of America, as
represented by the Secretary, Dept. of Health and Human
Services
Bethesda
MD
|
Family ID: |
60242757 |
Appl. No.: |
16/692849 |
Filed: |
November 22, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15297054 |
Oct 18, 2016 |
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16692849 |
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62332277 |
May 5, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/08 20130101;
A61K 38/58 20130101; A61K 38/1767 20130101; A61K 38/005 20130101;
A61K 38/10 20130101; A61K 47/545 20170801; A61K 47/60 20170801;
A61K 38/07 20130101 |
International
Class: |
A61K 38/00 20060101
A61K038/00; A61K 38/17 20060101 A61K038/17; A61K 38/58 20060101
A61K038/58; A61K 38/08 20060101 A61K038/08; A61K 38/10 20060101
A61K038/10; A61K 38/07 20060101 A61K038/07; A61K 47/60 20060101
A61K047/60; A61K 47/54 20060101 A61K047/54 |
Claims
1. A method, comprising administering to a subject a composition
comprising: TABLE-US-00006 (SEQ ID NO: 4) VitE-AAPV; (SEQ ID NO: 5)
VitE-PEG-AAPV; (SEQ ID NO: 6) VitE-PEG-AAPV-CMK; or (SEQ ID NO: 7)
VitE-AAPV-CMK.
2. The method of claim 1, wherein the vitamin E (VitE) is selected
from the group consisting of alpha-tocopherol, beta-tocopherol,
gamma-tocopherol, delta-tocopherol, alpha-tocotrienol,
beta-tocotrienol, gamma-tocotrienol, and delta-tocotrienol
3. The method of claim 1, wherein the composition further comprises
a pharmaceutically acceptable carrier.
4. The method of claim 3, wherein the pharmaceutically acceptable
carrier is a lipid.
5. The method of claim 1, wherein the composition is administered
in a liposome or reconstituted high density lipoprotein.
6. The method of claim 1, wherein the method is a method of
treating alpha-1-antitrypsin (A1AT) deficiency, hyperlipidemia,
atherosclerosis, restenosis, peripheral vascular disease, acute
coronary syndrome, reperfusion myocardial injury, asthma, chronic
pulmonary obstructive disorder, and/or emphysema.
7. The method of claim 1, wherein the composition is administered
to the subject by injection or infusion.
8. The method of claim 7, wherein the composition is administered
to the subject intravenously, subcutaneously, intra-arterially, or
intrapericardially.
9. The method of claim 1, wherein the composition is incorporated
in an implantable device.
10. The method of claim 9, wherein the implantable device is a
stent.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/297,054, filed Oct. 18, 2016, which in turn claims the
benefit of the earlier filing date of U.S. Provisional Application
No. 62/332,277, filed May 5, 2016. The entire contents of both
applications are incorporated herein by reference.
FIELD
[0002] This disclosure relates to lipoprotein-targeting compounds,
compositions, and systems employing a lipoprotein-targeting
molecule, such as a naturally associating protein or fragment
thereof, optionally included as part of a fusion molecule. Also
disclosed are methods of using such compounds, compositions, and
systems therapeutically, for instance to treat cardiovascular
disease or protein deficiency-related diseases and conditions.
BACKGROUND
[0003] Cardiovascular disease (CVD) is the major cause of morbidity
and mortality in developed countries and atherosclerosis is the
major cause of CVD. Accumulation of cholesterol in the arterial
wall and vascular inflammation are at the center of pathogenesis of
atherosclerosis. Treatments controlling delivery of cholesterol and
inflammation (statins) reduced incidence of CVD by 30-40%. There
is, however, an urgent need for further reduction.
[0004] A most promising direction is complementing reduction in
levels of the proatherogenic lipoproteins with increasing levels of
the anti-atherogenic lipoprotein, high density lipoprotein (HDL),
"HDL therapy". The success of HDL therapy depends on the method of
elevation of HDL. Presently, the most successful approach is direct
infusion of exogenous HDL. Infusion of reconstituted HDL (rHDL)
however has considerable limitations due to high cost and
requirement for intravenous delivery making it suitable mainly for
acute treatment.
[0005] Epidemiological studies have clearly identified elevated
plasma cholesterol as an independent risk factor for the
development of CVD (Kannel et al., Ann Intern Med 90: 85-91, 1979).
Plasma cholesterol is carried in emulsions of lipid and protein
called lipoproteins. Lipoproteins exist as a polydisperse
distribution of distinct particle classes most commonly classified
by density as very low, low, intermediate and high-density
lipoproteins. A likely overly simplistic but well accepted paradigm
for the role of lipoproteins in the development CVD is that excess
low density lipoproteins (LDL) promote CVD, by depositing
cholesterol in atherosclerotic plaque, whereas high density
lipoprotein (HDL) particles remove excess cholesterol and perhaps
mediate other anti-atherogenic effects. The primary metric for
assessment of CVD risk related to these lipoproteins is largely
based on the cholesterol content of each of these lipoprotein
particles (that is, LDL-C and HDL-C).
[0006] The major lipoprotein classes contain distinct subclasses,
with different physical and chemical properties and differ in their
relationship with CVD. For example, total LDL is composed of at
least two subclasses: large buoyant and small dense LDL, which is
particularly proatherogenic (Chapman et al., Eur Heart J 19 Suppl
A: A24-30, 1998). The subclass distribution of HDL is much more
complex; it consists of numerous distinct subclasses with varying
lipid and protein compositions. Modern mass spectrometry (MS)
techniques have allowed for thorough characterizations of the
lipoprotein proteomes of both LDL and HDL. While LDL typically
contains only a few prototypical proteins, such as apoB, apoE,
apoC's etc., HDL particles may contain as many as 90 different
proteins among its particle subclasses (Vaisar et al., J Clin
Invest 117: 746-756, 2007; Karlsson et al., Proteomics 5: 551-565,
2005; Karlsson et al., Proteomics 5: 1431-1445, 2005; Davidson, The
HDL Proteome Watch available online at
homepages.uc.edu.about.davidswm/HDLproteome.html, 2015; Gordon et
al., J Proteome Res 9: 5239-5249, 2010). This proteomic diversity
likely accounts for the dramatic functional diversity found in HDL,
including numerous potential mechanisms for protection against
inflammation and oxidation, as well as anti-coagulant and
pro-vasodilatory functions, to name only a few (Gordon et al.,
Trends Endocrinol Metab 22: 9-15, 2011).
[0007] Alpha-1-antitrypsin (A1AT) deficiency occurs in about 1 in
2500 individuals in the United States and Europe. People with this
condition develop severe liver disease and emphysema/chronic
obstructive pulmonary disease (COPD). The current treatment for
alpha-1-antitrypsin (A1AT) deficiency involves intravenous infusion
of purified human A1AT protein. This treatment strategy is very
expensive and only modestly effective. An improvement in A1AT
treatment effectiveness in a mouse model of emphysema has been
demonstrated by pre-incubating A1AT with high density lipoprotein
(HDL) particles prior to infusion. This resulted in improvements in
lung morphology and inflammatory markers in the lung compared to
A1AT treatment alone. The mechanism for this improvement in
function of A1AT when bound to HDL is believed to be increased
trafficking of A1AT to the lung.
SUMMARY
[0008] Described herein is the development, design and construction
of a class of lipoprotein targeting protease inhibitors. Peptides
with protease inhibitor activity (antiprotease peptides) are
conjugated to hydrophobic, lipoprotein targeting molecules using,
for instance, amine reactive chemistry. Methods of use of the
resultant lipoprotein targeting protease inhibitor (antiprotease)
molecules are also described.
[0009] There is provided herein in a first embodiment a lipoprotein
targeting protease inhibitor peptide having the generic structure:
T-I, in which T is a hydrophobic entity comprising a vitamin E
(VitE), an acyl chain, or cholesterol; and I is a peptide-based
protease inhibitor; where T is covalently attached directly to I,
or indirectly by way of a hydrophilic linker L (the latter
resulting in the generic structure: T-L-I).
[0010] The protease inhibitor component I can be any of myriad
peptide-based protease inhibitors (antiproteases), including
peptide-based inhibitors of elastase, matrix metalloprotease (MMP),
cathepsin, chymase, thrombin, coagulation factor IX, coagulation
factor X, urokinase-type plasminogen activator (uPA), tissue-type
plasminogen activator (tPA), and proteolytic components of the
complement cascade (C1r, C1s, MASPs 1-3, C2, Factor B, Factor D or
Factor I).
[0011] Optionally, the lipoprotein targeting protease inhibitor
peptide may comprise a linker L, for instance which is a
hydrophilic linker comprising polyethylene glycol (PEG) or
succinimide. Inhibitor peptides with different length linkers are
specifically contemplated.
[0012] Also provided herein are pharmaceutical compositions,
comprising at least one lipoprotein targeting protease inhibitor
peptide, and a pharmaceutically acceptable carrier. Optionally, the
peptide in such a composition is contained in or part of a
lipoprotein, such as a HDL. Methods comprising administering such a
pharmaceutical composition to a subject are also provided.
[0013] Also provided are methods of producing HDL with enriched
protease inhibitor (antiprotease) activity, comprising contacting
HDL with a lipoprotein targeting protease inhibitor peptide as
provided herein.
[0014] Yet additional embodiments are protease inhibitor enriched
HDL, comprising HDL and a lipoprotein targeting protease inhibitor
peptide as described herein. In examples of this embodiment, the
protease inhibitor enriched HDL reconstituted HDL (rHDL).
[0015] The foregoing and other features and advantages will become
more apparent from the following detailed description of several
embodiments, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1A-1C. Effect of rosuvastatin on plasma lipids and
lipoprotein particle numbers. (FIG. 1A) Rosuvastatin effects on
total plasma lipid levels (TC=total cholesterol; HDL-C=HDL
cholesterol; LDL-C=LDL cholesterol; TG=triglyceride). The effect of
rosuvastatin on LDL particle number (FIG. 1B) and HDL particle
number (FIG. 1C) were measured by nuclear magnetic resonance. T0
and T28 are time points indicating baseline and after 28 days of
rosuvastatin treatment, respectively. Data are mean.+-.standard
deviation. * indicates p<0.01.
[0017] FIG. 2. Effect of rosuvastatin on plasma lipid distributions
by size exclusion chromatography. Plasma from patients at baseline
(T0) and after 28 days (T28) of rosuvastatin treatment was
separated on two Superose 6 columns arranged in series. Collected
fractions were analyzed for total cholesterol, free cholesterol,
phosphatidylcholine and triglyceride. Data are mean.+-.standard
deviation.
[0018] FIG. 3. ApoB spectral counts correlate with LDL particle
number. As validation of the semi-quantitative potential of
spectral counting under our experimental conditions we compared
spectral counts for apolipoprotein B (apoB) vs. LDL particle
number. ApoB is a core protein of LDL and has a well-established
1:1 (mol apoB:mol LDL) stoichiometry.
[0019] FIG. 4A-4B. Rosuvastatin alters the lipoprotein proteome.
Statistically significant changes to the LDL (FIG. 4A) and HDL
(FIG. 4B) proteomes resulting from rosuvastatin treatment are
displayed as percent change compared to baseline. HDL-L=large HDL;
HDL-S=small HDL; PGRP-L=N-acetylmuramoyl-L-alanine amidase.
Statistical comparisons were made using student's T test. All
displayed data are p<0.05.
[0020] FIG. 5A-5C. Quantitative measurement of alpha-1-antitrypsin
on HDL and in plasma. (FIG. 5A) Individual patient spectral counts
for alpha-1-antitrypsin (A1AT) in large HDL at baseline (T0) and
after 28 days (T28) of rosuvastatin treatment, n=10 for each time
point. The "n=5" indicator points to data from 5 subjects with a
high degree of overlap. (FIG. 5B) Quantitative measurement of A1AT
in large HDL by ELISA assay. (FIG. 5C) Time course of plasma A1AT
concentrations during rosuvastatin treatment and after two-week
washout period (Day 42 time point). * indicates p<0.05 and **
indicates p<0.01 compared to T0.
[0021] FIG. 6A-6B. Structural prediction of lipid binding by
alpha-1-antitrypsin. (FIG. 6A) Predicted binding of
alpha-1-antitrypsin (A1AT) to a lipid surface (red spheres). Inset
demonstrates that methionine residues (Met 351 and Met 358) are
embedded in the lipid (white arrows) and indicates the cut site for
neutrophil elastase (red arrow). (FIG. 6B) Predicted lipid binding
of A1AT structure with the reactive center loop removed (A1AT
.DELTA. 346).
[0022] FIG. 7A-7B. Alpha-1-antitrypsin has reduced anti-elastase
activity when bound to reconstituted HDL. Reconstituted HDL (rHDL)
were prepared from apoA-I and phospholipids by cholate dialysis and
then co-incubated with alpha-1-antitrypsin (A1AT) to generate A1AT
enriched rHDL. (FIG. 7A) Size exclusion chromatography on tandem
Superdex 200 columns was used to isolate HDL bound A1AT from lipid
free protein. (FIG. 7B) The ability of lipid free and rHDL bound
A1AT to inhibit neutrophil elastase (NE) activity was measured by
fluorometric assay.
[0023] FIG. 8. Binding to HDL protects alpha-1-antitrypsin
anti-elastase activity from oxidation by H.sub.2O.sub.2. HDL
isolated from healthy human donors was co-incubated with
alpha-1-antitrypsin (A1AT) to generate A1AT enriched nHDL. Lipid
free A1AT and A1AT nHDL were exposed to varying concentrations of
H.sub.2O.sub.2 for 30 minutes before measurement of anti-elastase
activity by fluorometric assay. Nonlinear regression analysis was
used for comparison of curve fits and found the two curves to be
significantly different (p<0.0001).
[0024] FIG. 9A-9E. Alpha-1-antitrypsin enriched HDL prevents
elastase induced TNF-.alpha. production by macrophages. (FIG. 9A)
J774 mouse macrophages treated with increasing amounts of porcine
pancreatic elastase (PPE) or heat inactivated elastase for 4 hours,
TNF-.alpha. in the culture media was measured by ELISA. (FIG. 9B)
J774 cells pretreated with PBS, isolated human HDL (nHDL), the same
HDL enriched with alpha-1-antitrypsin (A1AT nHDL), or lipid free
A1AT for 1 hour prior to PPE addition. (FIG. 9C) The ability of
each of the cell treatments to inhibit elastase activity was
measured in a cell-free assay. (FIG. 9D) Treatments were
pre-incubated with PPE prior to addition to cells and TNF-.alpha.
was measured in the culture media after 4 hours. (FIG. 9E) J774
cells were pre-incubated with each treatment for 1 hour; cells were
then washed twice with PBS and placed in fresh media containing PPE
and TNF-.alpha. was measured in the culture media after 4 hours.
All experiments were repeated at least 3 times and were done in
triplicates. Treatments were compared using one-way ANOVA and
Tukey's multiple comparisons test, p<0.05 was considered
significant. The letters above each treatment indicate statistical
significance; within each graph, bars bearing different letters
were statistically different from each other.
[0025] FIG. 10A-10B. Elastase treatment of HDL. Enrichment of HDL
with A1AT protects HDL proteins (apoA-I and apoA-II) from
degradation by elastase, SDS-PAGE with Coomassie blue stain for
total protein (FIG. 10A). Additionally, elastase treatment results
in degradation of pre-beta HDL and this is also protected by A1AT,
one-dimensional native gel electrophoresis with western blot for
apoA-I (FIG. 10B).
[0026] FIG. 11A-11C. Functional analyses of elastase treated HDL.
The activity of three common HDL functions was measured in native
HDL or A1AT-HDL with or without treatment with elastase. For native
HDL, elastase treatment resulted in reduced cholesterol efflux
(FIG. 11A), PON1 activity (FIG. 11B), and LCAT activity (FIG. 11C).
A1AT-HDL was protected from elastase mediated reductions of all of
these functions.
[0027] FIG. 12 is a graph showing that peptides conjugated to
vitamin E bind HDL in plasma. The black line shows the FPLC elution
profile of cholesterol indicating the distribution pattern of
plasma lipoproteins in collected fractions. The red and green lines
represent the elution profile of a fluorescent peptide without
(red) or with (green) VitE conjugation. This data indicates that,
when conjugated to Vitamin E, small peptides preferentially bind to
HDL in human plasma.
[0028] FIG. 13A-13B. FIG. 13A is a schematic representation of an
example lipoprotein targeting protease inhibitor molecule,
comprising vitamin E (VitE) linked to the elastase-inhibitory
peptide AAPV-CMK (SEQ ID NO: 3). FIG. 13B is a drawing of how
lipoprotein targeting protease inhibitor molecules described
herein, exemplified by VitE-AAPV-CMK, interact with an HDL
particle.
[0029] FIG. 14 is an overview of how the exemplary lipoprotein
targeting protease inhibitor molecule VitE-AAPV is constructed from
two components, VitE-PEG.sub.2000-NHS (e.g., commercially available
from NANOCS Inc., Catalog No. PG2-NSVE-2k) and the elastase
inhibitor peptide, Ala-Ala-Pro-Val-CH.sub.2Cl (SEQ ID NO: 2), and
the resultant structure.
[0030] FIG. 15 is a series of three mass spectrometer traces,
showing the change of molecular weight of the compound during
reaction between VitE-PEG-NHS with the elastase inhibitor peptide,
AAPV-CH.sub.2Cl (SEQ ID NO: 2).
[0031] FIG. 16 is a graph showing elastase inhibitor activity of
the reactants AAPV (SEQ ID NO: 1) and VitE-PEG and the product
fusion peptide, E-AAPV (VitE-PEG.sub.2000-AAPV). This data shows
that VitE-PEG molecule alone does not convey elastase inhibition
and that Vitamin E conjugation to the AAPV peptide does not affect
elastase inhibition activity.
[0032] FIG. 17 is a graph showing that the E-AAPV peptide binds HDL
and confers dose-dependent elastase inhibitor activity. Isolated
Human HDL was co-incubated with the E-AAPV peptide for 30 minutes
at 37.degree. C. and then HDL was reisolated by FPLC to remove
unbound E-AAPV. The HDL was then tested for elastase inhibition
activity and compared to control HDL which was coincubated with PBS
(no peptide) and repurified. Samples were matched based on
phospholipid content.
SEQUENCE LISTING
[0033] The nucleic and/or amino acid sequences listed in the
accompanying Sequence Listing are shown using standard letter
abbreviations for nucleotide bases, and three letter code for amino
acids, as defined in 37 C.F.R. 1.822. The Sequence Listing is
submitted as an ASCII text file named Sequence Listing, created on
Nov. 22, 2019, .about.8 KB, which is incorporated by reference
herein.
[0034] SEQ ID NO: 1 is the antiprotease peptide
Ala-Ala-Pro-Val.
[0035] SEQ ID NO: 2 is the modified antiprotease peptide
Ala-Ala-Pro-Val-CH.sub.2Cl.
[0036] SEQ ID NO: 3 is the modified antiprotease peptide
Ala-Ala-Pro-Val-CMK.
[0037] SEQ ID NO: 4 is the lipoprotein targeting protease inhibitor
(antiprotease) molecule VitE-Ala-Ala-Pro-Val.
[0038] SEQ ID NO: 5 is the lipoprotein targeting protease inhibitor
(antiprotease) molecule VitE-PEG-Ala-Ala-Pro-Val.
[0039] SEQ ID NO: 6 is the lipoprotein targeting protease inhibitor
(antiprotease) molecule VitE-PEG-Ala-Ala-Pro-Val-CMK. Different
versions of this molecule are contemplated and explicitly provided
herein, with different length PEG moieties.
[0040] SEQ ID NO: 7 is the lipoprotein targeting protease inhibitor
(antiprotease) molecule VitE-Ala-Ala-Pro-Val-CMK.
[0041] SEQ ID NO: 8 is the lipoprotein targeting protease inhibitor
(antiprotease) molecule VitE-PEG-KRCCPDTCGIKCL. Different versions
of this molecule are contemplated and explicitly provided herein,
with different length PEG moieties
[0042] SEQ ID NO: 9 is the lipoprotein targeting protease inhibitor
(antiprotease) molecule VitE-PEG-KRMMPDTMGIKML. Different versions
of this molecule are contemplated, with different length PEG
moieties
[0043] SEQ ID NO: 10 is the lipoprotein targeting protease
inhibitor (antiprotease) molecule VitE-PEG-EEIIMD. Different
versions of this molecule are contemplated, with different length
PEG moieties SEQ ID NO: 11 is the thrombin and/or coagulation
factors IX and X inhibitor peptide Hirudin
TABLE-US-00001 (MTYTDCTESGQNLCLCEGSNVCGQGNKCILGSDGEKNQCVTGEGTPKP
QSHNDGDFEEIPEEYLQ).
[0044] SEQ ID NO: 12 is the plasminogen inhibitor peptide
aprotinin
TABLE-US-00002 (RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSA
EDCMRTCGGA).
[0045] SEQ ID NO: 13 is the lipoprotein targeting protease
inhibitor (antiprotease) molecule
VitE-(PEG).sub.2-Lys-Gly-Ser-Gly-Ala-Ala-Pro-Val-CMK
(VitE-PEG-KGSGAAPV-CMK), which serves as a non-fluorescent
equivalent to SEQ ID NO: 14. Different versions of this molecule
are contemplated, with different length PEG moieties
[0046] SEQ ID NO: 14 is the fluorescently labeled lipoprotein
targeting protease inhibitor (antiprotease) molecule
VitE-(PEG).sub.2-Lys[FITC]-Gly-Ser-Gly-Ala-Ala-Pro-Val-CMK
(VitE-PEG-K.sup.(F)GSGAAPV-CMK). Different versions of this
molecule are contemplated, with different length PEG moieties.
[0047] SEQ ID NO: 15 is a peptide inhibitor of (leukocyte) elastase
R.sub.1-W.sub.p-X.sub.n-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-Y.sub.m-R.sub-
.2 in which AA.sub.1: -Arg-, -Phg- and -Nle- or is a bond;
AA.sub.2: -Ala-, -Phg-, -Cit- and -Nle-; AA.sub.3: -Trp-, -Val- and
-Tyr-; AA.sub.4: -Phg- and -Gly-; W, X and Y are independently
selected from the group consisting of coded or uncoded amino acids;
p, n and m range between 0 and 1; R.sub.1 is selected from the
group consisting of H, substituted or unsubstituted non-cyclic
aliphatic group, substituted or unsubstituted alicyclyl,
substituted or unsubstituted heterocyclyl, substituted or
unsubstituted heteroarylalkyl, substituted or unsubstituted aryl,
substituted or unsubstituted aralkyl and R.sub.5--CO--; R.sub.2 is
selected from the group consisting of --NR.sub.3R4, --OR3 and
--SR.sub.3; wherein R.sub.3 and R.sub.4 are independently selected
from the group consisting of H, substituted or unsubstituted
non-cyclic aliphatic group, substituted or unsubstituted alicyclyl,
substituted or unsubstituted heterocyclyl, substituted or
unsubstituted heteroarylalkyl, substituted or unsubstituted aryl
and substituted or unsubstituted aralkyl; wherein R.sub.5 is
selected from the group consisting of H, substituted or
unsubstituted non-cyclic aliphatic group, substituted or
unsubstituted alicyclyl, substituted or unsubstituted aryl,
substituted or unsubstituted aralkyl, substituted or unsubstituted
heterocyclyl and substituted or unsubstituted heteroarylalkyl; and
provided that when AA.sub.1 is a bond, AA.sub.2 is -Phg- and
AA.sub.3 is -Trp-. R.sub.1 and R.sub.2 groups are bound to the
amino-terminal (N-terminal) and carboxy-terminal (C-terminal) of
the peptide sequences respectively.
DETAILED DESCRIPTION
I. Abbreviations
[0048] A1AT alpha-1-antitrypsin
[0049] CMK chloromethylketone
[0050] CVD cardiovascular disease
[0051] E-AAPV vitamin E fused to the antiprotease peptide AAP, with
or without a linker
[0052] FDR false discovery rate
[0053] FPLC fast protein liquid chromatography
[0054] HDL high density lipoprotein
[0055] I protease inhibitor (antiprotease) moiety
[0056] L hydrophilic linker
[0057] LCAT lecithin cholesterol acyltransferase
[0058] LDL low density lipoprotein
[0059] NE neutrophil elastase
[0060] NHS N-hydroxysuccinimide
[0061] nHDL native HDL
[0062] PPE porcine pancreatic elastase
[0063] PVD peripheral vascular disease
[0064] RCL reactive center loop
[0065] rHDL reconstituted HDL
[0066] SERPIN serine protease inhibitor
[0067] T hydrophobic, lipoprotein targeting moiety
[0068] TNF-.alpha. tumor necrosis factor alpha
[0069] VLDL very low density lipoprotein
[0070] VitE vitamin E (also, in some instances, simply "E")
II. Terms
[0071] 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).
[0072] In order to facilitate review of the various embodiments of
the invention, the following explanations of specific terms are
provided:
[0073] Alpha-1-antitrypsin (A1AT): A protease inhibitor belonging
to the SERPIN (serum trypsin inhibitor) superfamily. Alpha
1-antitrypsin is also referred to as alpha-1 proteinase inhibitor
(A1PI) because it inhibits a wide variety of proteases. It protects
tissues from enzymes of inflammatory cells, especially neutrophil
elastase; it also inhibits plasmin, thrombin, trypsin,
chymotrypsin, and plasminogen activator. In its absence (such as in
A1AT deficiency), neutrophil elastase is free to break down
elastin, which contributes to the elasticity of the lungs,
resulting in respiratory complications such as emphysema, or COPD
(chronic obstructive pulmonary disease) in adults and cirrhosis in
adults or children. Synonyms: SERPINA1, serpin peptidase inhibitor,
AAT, PI, PI1, A1A, PRO2275.
[0074] Analog, derivative or mimetic: An analog is a molecule that
differs in chemical structure from a parent compound, for example a
homolog (differing by an increment in the chemical structure, such
as a difference in the length of an alkyl chain), a molecular
fragment, a structure that differs by one or more functional
groups, a change in ionization. Structural analogs are often found
using quantitative structure activity relationships (QSAR), with
techniques such as those disclosed in Remington (The Science and
Practice of Pharmacology, 19th Edition (1995), chapter 28). A
derivative is a biologically active molecule derived from the base
structure. A mimetic is a molecule that mimics the activity of
another molecule, such as a biologically active molecule.
Biologically active molecules can include chemical structures that
mimic the biological activities of a compound.
[0075] Animal: Living multi-cellular vertebrate organisms, a
category that includes, for example, mammals and birds. The term
mammal includes both human and non-human mammals. Similarly, the
term "subject" includes both human and veterinary subjects, for
example, humans, non-human primates, dogs, cats, mice, rates,
rabbits, horses, and cows.
[0076] Apolipoprotein A-I (apoA-I): A major protein component of
high density lipoprotein (HDL) complex in plasma. Apolipoprotein
A-I can promote cholesterol efflux from tissues and transport to
the liver for excretion. It is a cofactor for lecithin cholesterol
acyltransferase (LCAT) which is responsible for the formation of
most plasma cholesteryl esters. In addition, apoA-I has many other
pleiotropic effects, such as anti-inflammatory, anti-thrombotic,
and improving insulin sensitivity, which mechanistically are not
understood but may contribute to the anti-atherogenic effect of
HDL.
[0077] In particular examples, an apoA-I protein, fragment or
variant thereof is capable of promoting cholesterol efflux. For
example, an apoA-I protein, fragment or variant thereof is
administered to a subject to promote cholesterol efflux. Unless the
context clearly indicates otherwise, the term apoA-I includes any
apoA-I gene, cDNA, mRNA, or protein from any organism and is
capable of promoting cholesterol efflux.
[0078] Nucleic acid and protein sequences for apoA-I are publicly
available. For example, GenBank Accession Nos. NM_144772.2 (human)
and NM_009692 (mouse) disclose an apoA-I nucleic acid sequence, and
GenBank Accession Nos. NP_658985 (human), AAB21444 (bovine) and
NP_033822 (mouse) disclose apoA-I protein sequences, all of which
are incorporated by reference as provided by GenBank on May 3,
2016.
[0079] Apolipoprotein C-II (apoC-II): A 79 amino acid protein,
which plays a role in plasma lipid metabolism as an activator of
lipoprotein lipase (LPL). This protein includes three amphipathic
helices: helix 1, residues 16-38; helix 2, residues 45-58; and
helix 3, residues 64-74. The lipase-activating region of apoC-II
has previously been localized to the C-terminal domain of the
sequence, from about residue 56, whereas the N-terminal domain
(residues 1-50) of the sequence is involved in lipid binding.
[0080] Unless the context clearly indicates otherwise, the term
apoC-II includes any apoC-II gene, cDNA, mRNA, or protein from any
organism and is capable of activating lipoprotein lipase. Nucleic
acid and protein sequences for apoC-II are publicly available. For
example, GenBank Accession No. NM_009695 (human) discloses an
apoC-II nucleic acid sequence, and GenBank Accession Nos. AAH05348
(human), NP_001078821 (rat), NP_001095850 (bovine), and NP_033825
(mouse) disclose additional apoC-II protein sequences, all of which
are incorporated by reference as provided by GenBank on May 3,
2016.
[0081] Atherosclerosis: The progressive narrowing and hardening of
a blood vessel over time. Atherosclerosis is a common form of
arteriosclerosis in which deposits of yellowish plaques (atheromas)
containing cholesterol, lipoid material and lipophages are formed
within the intima and inner media of large and medium-sized
arteries. Treatment of atherosclerosis includes reversing or
slowing the progression of atherosclerosis, for example as measured
by the presence of atherosclerotic lesions and/or functional signs
of the disease, such as improvement in cardiovascular function as
measured by signs (such as peripheral capillary refill), symptoms
(such as chest pain and intermittent claudication), or laboratory
evidence (such as that obtained by EKG, angiography, or other
imaging techniques).
[0082] Cardiovascular: Pertaining to the heart and/or blood
vessels.
[0083] Cardiovascular disease (CVD): A group of diseases that
includes, but is not limited to, angina pectoris (commonly known as
"angina"), arteriolosclerosis, atherosclerosis (ASCVD),
cerebrovascular disease (such as stroke), intermittent
claudication, congestive heart failure, coronary artery disease
(CAD), coronary insufficiency, elevated cholesterol, ischemic heart
disease, myocardial infarction, peripheral vascular disease, small
vessel disease, thrombosis, transient ischemic attack, and
hypertension. Atherosclerosis usually results from the accumulation
of fatty material, inflammatory cells, extracellular matrices and
plaque. Clinical symptoms and signs indicating the presence of CVD
may include one or more of the following: chest pain and other
forms of angina, shortness of breath, sweatiness, Q waves or
inverted T waves on an EKG, a high calcium score by CT scan, at
least one stenotic lesion on coronary angiography, and heart
attack. Subclinical ASCVD can be identified by imaging tests (such
as CT measures of coronary calcification, or MRI measures of
coronary or aortic plaque, and/or ultrasound evidence of carotid
plaque or thickening).
[0084] Cholesterol absorption inhibitor: A class of cholesterol
lowering drugs that block absorption of cholesterol at the brush
border of the intestine without affecting absorption of
tri-glycerides or fat soluble vitamins. These drugs are not
systemically absorbed and can lower cholesterol on their own (i.e.
without the use of additional drugs). An exemplary cholesterol
absorption inhibitor is ezetimibe (EZETROL.TM.).
[0085] Cholesterol lowering agent: An agent that lowers the level
of cholesterol in a subject, such as a pharmaceutical, vitamin, or
small molecule. One of skill in the art can readily identify
assays, such as blood screening, to determine the effect of
cholesterol. Agents include, but are not limited to, niacin, the
statins (e.g., ZOCOR.TM. LIPITOR.TM., PRAVACOL.TM., LESCOR.TM.,
MEVACOR.TM.), bile acid binding resins (e.g., QUESTRAN.TM.), and
fibrates (e.g. LOPID.TM., LIPIDIL MICRO.TM.).
[0086] Complex (complexed): Two compounds/molecules (e.g., two
proteins, a protein and a lipid; a protein and a lipid particle,
etc.), or fragments or derivatives thereof, are said to form a
complex when they measurably associate with each other in a
specific manner. Such association can be measured in any of various
ways, both direct and indirect. Direct methods may include
co-migration in non-denaturing fractionation conditions, for
instance. Indirect measurements of association will depend on
secondary effects caused by the association of the two components
in the complex. Representative methods for detecting,
characterizing, and measuring formation of certain complex(es) are
presented herein; additional methods will be recognized by those of
ordinary skill in the relevant art(s).
[0087] Coronary Artery Disease: In coronary artery disease, the
coronary arteries become narrowed (stenosed) or blocked (occluded)
by a gradual build-up of fat (cholesterol) within or on the artery
wall, which reduces blood flow to the heart muscle. This build-up
is called atherosclerotic plaque or simply plaque.
[0088] If plaque narrows the lumen or channel of the artery, it may
make it difficult for adequate quantities of blood to flow to the
heart muscle. If the build-up reduces flow only mildly, there may
be no noticeable symptoms at rest, but symptoms such as chest
pressure may occur with increased activity or stress. Other
symptoms include heartburn, nausea, vomiting, shortness of breath
and heavy sweating.
[0089] When flow is significantly reduced and the heart muscle does
not receive enough blood flow to meet its needs (cardiac ischemia),
severe symptoms such as chest pain (angina pectoris), heart attack
(myocardial infarction), or rhythm disturbances (arrhythmias) may
occur. A heart attack usually is the result of a completely blocked
artery, which may damage the heart muscle.
[0090] There are three conventional ways to treat atherosclerotic
disease: medication, surgery, and minimally invasive interventional
procedures such as stent implantation, percutaneous transluminal
coronary angioplasty (PTCA), intravascular radiotherapy,
atherectomy and excimer laser. The purpose of these treatments is
to eliminate or reduce atherosclerotic narrowing of the coronary
blood vessels and hence eliminate or reduce symptoms, and in the
case of coronary artery disease, decrease the risk of heart
attack.
[0091] Domain: A domain of a protein or other molecule is a part of
the molecule that shares common structural, physiochemical and
functional features; for example hydrophobic, polar, globular,
helical domains or properties, for example a DNA binding domain, an
ATP binding domain, lipoprotein lipase activating domain, a
membrane-inserting domain, and the like. In a particular example, a
fusion molecule includes a first domain and a second domain (though
they can occur in any order) one of which is hydrophobic and
capable of associating with (and/or specifically targeted to) a
lipoprotein (such as HDL or LDL), while the other domain ha
protease activity. In embodiments described herein, the first and
second domains are joined covalently to each other by way of a
linker.
[0092] Dyslipidemic disorder: A disorder associated with any
altered amount of any or all of the lipids or lipoproteins in the
blood. Dyslipidemic disorders include, for example, hyperlipidemia,
hyperlipoproteinemia, hypercholesterolemia, hypertriglyceridemia,
HDL deficiency, apoA-I deficiency, and cardiovascular disease
(i.e., coronary artery disease, atherosclerosis and
restenosis).
[0093] Heart failure (HF): The physiological state in which cardiac
output is insufficient in meeting the needs of the body and lungs.
This condition is also called "congestive heart failure," and is
most commonly caused when cardiac output is low and the lungs
become congested with fluid due to an inability of heart output to
properly match venous return. Heart failure can also occur in
situations of high output, where the ventricular systolic function
is normal but the heart can't process the augmentation of blood
volume. This can occur in overload situation (blood or serum
infusions), renal diseases, chronic severe anemia, beriberi
(vitamin B.sub.1/thiamine deficiency), thyrotoxicosis, Paget's
disease, arteriovenous fistulae, or arteriovenous malformations.
Heart failure includes left sided failure and right sided failure,
wherein the left and right ventricles are affected, respectively,
and biventricular failure. Ischemic heart disease (including
myocardial infarction), cigarette smoking, hypertension, obesity,
diabetes, and valvular heart disease are associated with increased
risk of heart failure. Viral myocarditis, human immunodeficiency
virus infections, connective tissue disease (such as systemic lupus
erythematous), drug (cocaine) abuse, and some chemotherapeutic
agents can cause heart failure.
[0094] High density lipoprotein (HDL): A class of heterogeneous
lipoproteins containing lipid and protein characterized by high
density (>1.063 g/mL) and small size (Stoke's diameter=5 to 17
nm). The various HDL subclasses vary in quantitative and
qualitative content of lipids, apolipoproteins, enzymes, and lipid
transfer proteins, resulting in differences in shape, density,
size, charge, and antigenicity. Apolipoprotein A-I (Apo-AI) is the
predominant HDL protein, although other apolipoproteins such as
Apo-AII and those referenced in the HDL Proteome Watch (Davidson,
The HDL Proteome Watch. available online at
homepages.uc.edu/.about.davidswm/HDLproteome.html, 2015) may be
present.
[0095] Epidemiological and clinical studies have established an
inverse association between levels of high-density lipoprotein
cholesterol (HDL-C) and risk of cardiovascular disease. More
particularly, clinical administration of reconstituted HDL (rHDL)
formulations has been shown to confer beneficial effects to
hypercholesterolemic patients suffering from recent acute coronary
syndromes (ACS).
[0096] HDL can be isolated by a number of different methods,
including for instance ultracentrifugation (e.g., double-step
ultracentrifugation in a potassium bromide (KBr) density gradient,
interval of 1.063-1.210 g/ml) and immunosorption (e.g., using
anti-Apo A-I column prepared by crosslinking polyclonal antibodies
directed against Apo A-I to Sepharose beads or another column
matrix). Methods of isolating natural/native HDL are within the
ability of an ordinarily skilled artisan.
[0097] Alternatively, HDL can be synthesized using defined
components, to provide "reconstituted" or "synthetic" HDL.
Typically, reconstituted HDL formulations comprise a protein such
as Apo-AI, a lipid such as phosphatidylcholine, and a detergent
such as cholate or deoxycholate. In addition, cholesterol or other
lipids may be included; synthetic or naturally-occurring lipids, or
combinations thereof, are appropriate. Additional proteins may also
be included; they may be isolated from natural or engineered
biological sources, or chemically synthesized. As discussed in U.S.
Pat. No. 5,652,339 (which is hereby incorporated by reference in
its entirety), it may be advantageous to produce reconstituted HDL
formulations without using organic solvents, which in some cases
are used for dissolving the lipid component when producing rHDL
formulation.
[0098] Injectable composition: A pharmaceutically acceptable fluid
composition comprising at least one active ingredient, e.g. a
lipoprotein targeted compound, such A1AT or a lipoprotein targeting
protease inhibitor fusion molecule. The active ingredient is
usually dissolved or suspended in a physiologically acceptable
carrier, and the composition can additionally comprise minor
amounts of one or more non-toxic auxiliary substances, such as
emulsifying agents, preservatives, and pH buffering agents and the
like. Such injectable compositions that are useful for use with the
fusion proteins of this invention are conventional; formulations
are well known in the art.
[0099] Inhibiting or treating a disease: Inhibiting the full
development of a disease, disorder or condition, for example, in a
subject who is at risk for a disease such as atherosclerosis and
cardiovascular disease. "Treatment" refers to a therapeutic
intervention that ameliorates a sign or symptom of a disease or
pathological condition after it has begun to develop. As used
herein, the term "ameliorating," with reference to a disease,
pathological condition or symptom, refers to any observable
beneficial effect of the treatment. The beneficial effect can be
evidenced, for example, by a delayed onset of clinical symptoms of
the disease in a susceptible subject, a reduction in severity of
some or all clinical symptoms of the disease, a slower progression
of the disease, a reduction in the number of relapses of the
disease, an improvement in the overall health or well-being of the
subject, or by other parameters well known in the art that are
specific to the particular disease.
[0100] Implant: A support device. For example, an implant is a
device that is employed to enhance and support existing passages,
channels, and conduits such as the lumen of a blood vessel. In an
example, an implant is an endovascular support. In a particular
example, an implant is a stent. In one example, an implant is
effective to maintain a vessel open. In the present disclosure, an
implant can be coated with or impregnated with one or more of the
disclosed peptides to assist with the treatment of a dyslipidemic
or vascular disorder.
[0101] Isolated: An "isolated" biological component (such as a
nucleic acid molecule, protein or organelle) is one that has been
substantially separated or purified away from other biological
components in the cell of the organism in which the component
naturally occurs, i.e., other chromosomal and extra-chromosomal DNA
and RNA, proteins and organelles. Nucleic acids and proteins that
have been "isolated" include nucleic acids and proteins purified by
standard purification methods. The term also embraces nucleic acids
and proteins prepared by recombinant expression in a host cell as
well as chemically synthesized nucleic acids.
[0102] Linker: A molecule that joins two other molecules, either
covalently, or through ionic, van der Waals or hydrogen bonds. In
particular examples, a linker comprises polyethylene glycol (PEG),
or succinimide, or another hydrophilic compound.
[0103] Lipid: A class of water-insoluble, or partially water
insoluble, oily or greasy organic substances, that are extractable
from cells and tissues by nonpolar solvents, such as chloroform or
ether. The most abundant kinds of lipids are fats or
triacylglycerols, which are major fuels for most organisms. Another
class of lipids is the polar lipids, which are major components of
cell membranes. The following table (Table 1) provides one way of
grouping major types of lipids; these have been grouped according
to their chemical structure:
TABLE-US-00003 TABLE 1 Lipid type Representative examples or
sub-groups Triacylglycerols Waxes Phosphoglycerides
phosphatidylethanolamine phosphatidylcholine phosphatidylserine
phosphatidylinositol cardiolipin Sphingolipids sphingomyelin
cerebrosides gangliosides Sterols and their (see Table 3) fatty
acid esters
[0104] Lipids and related molecules may also be broken down into
other recognized classes, such as those shown in Table 2:
TABLE-US-00004 TABLE 2 Scientific Name Abbreviation
Lyso-Phosphatidylcholine LY Sphingomyelin SP Phosphatidylcholine PC
Phosphatidylserine PS Phosphatidylinositol PI
Phosphatidylethanolamine PE Phosphatidylglycerol PG Cardiolipin CL
Free Fatty Acids FFA Monoacylglycerides MAG Diacylglycerides DAG
Triacylglycerides TAG Cholesterol Esters CE
[0105] Also included in the term lipid are the compounds
collectively known as sterols. Table 3 shows representative
sterols.
TABLE-US-00005 TABLE 3 Molecular Scientific Name Formula Common
Name 5b-cholestan-3b-ol C.sub.27H.sub.48O coprostanol
5a-cholestan-3b-ol C.sub.27H.sub.48O dihydrocholesterol
5-cholesten-3b-ol C.sub.27H.sub.46O cholesterol
5,24-cholestadien-3b-ol C.sub.27H.sub.44O desmosterol
5-cholestan-25a-methyl-3b-ol C.sub.28H.sub.42O campesterol
5-cholestan-24b-methyl-3b-ol C.sub.28H.sub.42O
dihydrobrassicasterol 5-cholesten-24b-ethyl-3b-ol C.sub.29H.sub.50O
b-sitosterol 5,22-cholestadien-24b-ethyl-3b-ol C.sub.29H.sub.48O
stigmasterol
[0106] In specific embodiments, the lipids are functional,
biologically active component(s) of naturally-occurring HDL or of
reconstituted high density lipoprotein (rHDL). Such lipids include
phospholipids, cholesterol, cholesterol-esters, fatty acids and/or
triglycerides. Preferably, the lipid is a phospholipid.
Non-limiting examples of phospholipids include phosphatidylcholine
(PC) (lecithin), sphingosine-1-phosphate (S1P), phosphatidic acid,
phosphatidylethanolamine (PE) (cephalin), phosphatidylglycerol
(PG), phosphatidylserine (PS), phosphatidylinositol (PI) and
sphingomyelin (SM) or natural or synthetic derivatives thereof.
Natural derivatives include egg PC, egg PG, soy bean PC,
hydrogenated soy bean PC, soy bean PG, brain PS, sphingolipids,
brain SM, galactocerebroside, gangliosides, cerebrosides, cephalin,
cardiolipin, and dicetylphosphate. Synthetic derivatives include
dipalmitoylphosphatidylcholine (DPPC),
didecanoylphosphatidylcholine (DDPC), dierucoylphosphatidylcholine
(DEPC), dimyristoylphosphatidylcholine (DMPC),
distearoylphosphatidylcholine (DSPC), dilaurylphosphatidylcholine
(DLPC), palmitoyloleoylphosphatidylcholine (POPC),
palmitoylmyristoylphosphatidylcholine (PMPC),
palmitoylstearoylphosphatidylcholine (PSPC),
dioleoylphosphatidylcholine (DOPC),
dioleoylphosphatidylethanolamine (DOPE),
dilauroylphosphatidylglycerol (DLPG),
distearoylphosphatidylglycerol (DSPG),
dimyristoylphosphatidylglycerol (DMPG),
dipalmitoylphosphatidylglycerol (DPPG),
distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol
(DOPG), palmitoyloleoylphosphatidylglycerol (POPG),
dimyristoylphosphatidic acid (DMPA), dipalmitoylphosphatidic acid
(DPPA), distearoylphosphatidic acid (DSPA),
dimyristoylphosphatidylethanolamine (DMPE),
dipalmitoylphosphatidylethanolamine (DPPE),
dimyristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine
(DPPS), distearoylphosphatidylethanolamine (DSPE),
dioleoylphosphatidylethanolamine (DOPE) dioleoylphosphatidylserine
(DOPS), dipalrnitoylsphingomyelin (DPSM) and
distearoylsphingomyelin (DSSM). The phospholipid can also be a
derivative or analogue of any of the above phospholipids.
[0107] Lipoprotein: A biochemical assembly that contains both
proteins and lipids, bound to the proteins, which allow fats to
move through the water inside and outside cells. There are five
major groups of lipoprotein particles, which, in order of molecular
size, largest to smallest, are chylomicrons, very low-density
lipoprotein (VLDL), intermediate-density lipoprotein (IDL),
low-density lipoprotein (LDL), and HDL. HDL contains the highest
proportion of protein to cholesterol; its most abundant
apolipoproteins are apo A-I and apo A-II. LDL contains
apolipoprotein B, and has a core consisting of linoleate and
includes esterified and non-esterified cholesterol molecules. LDL
particles are approximately 22 nm in diameter and have a mass of
about 3 million Daltons. Lipoprotein a, (Lp(a)) is a lipoprotein
subclass; lipoprotein a consists of an LDL-like particle and the
specific apolipoprotein(a) [apo(a)], which is covalently bound to
the apolipoprotein B of the LDL like particle.
[0108] Operably linked: A first nucleic acid sequence is operably
linked with a second nucleic acid sequence when the first nucleic
acid sequence is placed in a functional relationship with the
second nucleic acid sequence. For instance, a promoter is operably
linked to a coding sequence if the promoter affects the
transcription or expression of the coding sequence. Generally,
operably linked DNA sequences are contiguous and, where necessary
to join two protein-coding regions, in the same reading frame.
[0109] Peripheral Vascular Disease (PVD): A condition in which the
arteries and/or veins that carry blood to and from the arms, legs,
soft tissues and vital organs of the body, including the heart and
brain, become narrowed or occluded. This interferes with the normal
flow of blood, sometimes causing pain but often causing no readily
detectable symptoms. With progression of PVD, significant loss of
blood flow to tissue and organs can lead to tissue death, necrosis
and organ death.
[0110] The most common cause of PVD is atherosclerosis, a gradual
process in which cholesterol and scar tissue build up, forming
plaques that occlude the blood vessels. In some cases, PVD may be
caused by blood clots that lodge in the arteries and restrict blood
flow.
[0111] PVD affects about one in 20 people over the age of 50, or 8
million people in the United States. More than half the people with
PVD experience leg pain, numbness or other symptoms, but many
people dismiss these signs as a normal part of aging and do not
seek medical help.
[0112] The most common symptom of PVD is painful cramping in the
leg or hip, particularly when walking. This symptom, also known as
claudication, occurs when there is not enough blood flowing to the
leg muscles during exercise, such that ischemia occurs. The pain
typically goes away when the muscles are rested.
[0113] Other symptoms may include numbness, tingling or weakness in
the leg. In severe cases, people with PVD may experience a burning
or aching pain in an extremity such as the foot or toes while
resting, or may develop a sore on the leg or foot that does not
heal. People with PVD also may experience a cooling or color change
in the skin of the legs or feet, or loss of hair on the legs. In
extreme cases, untreated PVD can lead to gangrene, a serious
condition that may require amputation of a leg, foot or toes.
People with PVD are also at higher risk for heart disease and
stroke.
[0114] Typically most symptomatic PVD is ascribed to peripheral
artery disease (PAD) denoting the above described pathology
predominantly in arteries. The term PVD includes this symptomology
and pathology in all classes of blood vessels.
[0115] Polyethylene glycol (PEG) and PEG linkers: Polyethylene
glycol [structurally, poly(ethylene) glycol] is a chemical compound
composed of repeating ethylene glycol units. PEG is a typically
biologically inert, non-immunogenic chemical that confers greater
water solubility to proteins, labeling tags and crosslinkers into
which it is incorporated as constituent chemical group. Depending
on how the constituent monomer or parent molecule(s) are defined
(as ethylene glycol, ethylene oxide or oxyethylene), PEG compounds
are also known as PEO (polyethylene oxide) and POE
(polyoxyethylene). Purified PEG is most commonly available
commercially as mixtures of different oligomer sizes in broadly or
narrowly defined molecular weight (MW) ranges. For example, "PEG
600" typically denotes a preparation that includes a mixture of
oligomers having an average MW of 600. Likewise, "PEG 10000"
denotes a mixture of PEG molecules (n=195 to 265) having an average
MW of 10,000 g/mol.
[0116] The wide selection of commercially available crosslinking
reagents includes those that contain discrete-length polyethylene
glycol spacers. Such PEG groups increase reagent and conjugate
solubility, minimize toxic and immunological effects compared to
non-PEG spacers, and provide several options for accommodating
specific crosslinking distances. Commercially available PEG
compounds include: Amine-reactive Pegylation Reagents (which
contain an NHS ester group at one end); Amine-reactive Pegylated
Crosslinkers; Sulfhydryl-reactive Pegylated Crosslinkers; and
Bifunctional Pegylated Crosslinkers (e.g.,
amine-to-sulfhydryl linkers that contain an NHS ester at one end
and a maleimide group at the other).
[0117] Peptide: A polymer in which the monomers are amino acid
residues which 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 terms "peptide" or
"polypeptide" as used herein are intended to encompass any amino
acid sequence and include modified sequences such as glycoproteins.
The term "peptide" is specifically intended to cover naturally
occurring peptides, as well as those which are recombinantly or
synthetically produced. The term "residue" or "amino acid residue"
includes reference to an amino acid that is incorporated into a
peptide, polypeptide, or protein.
[0118] Pharmaceutically acceptable carriers: The pharmaceutically
acceptable carriers useful in this invention are conventional.
Remington's Pharmaceutical Sciences, by E. W. Martin, Mack
Publishing Co., Easton, Pa., 15th Edition (1975), describes
compositions and formulations suitable for pharmaceutical delivery
of the fusion proteins herein disclosed.
[0119] In general, the nature of the carrier will depend on the
particular mode of administration being 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, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
[0120] Phospholipid: A phospholipid consists of a water-soluble
polar head, linked to two water-insoluble non-polar tails (by a
negatively charged phosphate group). Both tails consist of a fatty
acid, each about 14 to about 24 carbon groups long. When placed in
an aqueous environment, phospholipids form a bilayer or micelle,
where the hydrophobic tails line up against each other. This forms
a membrane with hydrophilic heads on both sides. A phospholipid is
a lipid that is a primary component of animal cell membranes.
[0121] Purified: The term purified does not require absolute
purity; rather, it is intended as a relative term. Thus, for
example, a purified compound preparation is one in which the
compound is more enriched than the compound is in its generative
environment, for instance within a cell or in a biochemical
reaction chamber. In some embodiments, a preparation of compound is
purified such that the compound represents at least 50% of the
content of the preparation.
[0122] Recombinant: A recombinant nucleic acid molecule is one that
has a sequence that is not naturally occurring or has a sequence
that is made by an artificial combination of two otherwise
separated segments of sequence. This artificial combination can be
accomplished by chemical synthesis or, more commonly, by the
artificial manipulation of isolated segments of nucleic acids,
e.g., by genetic engineering techniques.
[0123] Similarly, a recombinant protein is one encoded for by a
recombinant nucleic acid molecule.
[0124] Serpins: A superfamily of proteins with similar structures
that were first identified for their protease inhibition activity.
The acronym serpin was originally coined because the first serpins
to be identified act on chymotrypsin-like serine proteases (serine
protease inhibitors). They have an unusual mechanism of action:
they irreversibly inhibit their target protease by undergoing a
large conformational change to disrupt its active site. This
contrasts with the more common competitive mechanism for protease
inhibitors that bind to and block access to the protease active
site. Protease inhibition by serpins controls an array of
biological processes, including coagulation and inflammation, and
consequently these proteins are the target of medical research.
[0125] Most serpins are protease inhibitors, targeting
extracellular, chymotrypsin-like serine proteases. These proteases
possess a nucleophilic serine residue in a catalytic triad in their
active site. Examples include thrombin, trypsin, and human
neutrophil elastase. Serpins act as irreversible, suicide
inhibitors by trapping an intermediate of the protease's catalytic
mechanism. Although most serpins control proteolytic cascades, some
proteins with a serpin structure are not enzyme inhibitors, but
instead perform diverse functions such as storage (as in egg
white--ovalbumin), transport as in hormone carriage proteins
(thyroxine-binding globulin, cortisol-binding globulin), and
molecular chaperoning (HSP47). The term serpin generally is used to
describe these members as well, despite their non-inhibitory
function, since they are evolutionarily related.
[0126] Statin: Any of a class of lipid-lowering drugs that reduce
serum cholesterol levels by inhibiting a key enzyme involved in the
biosynthesis of cholesterol, namely HMG-CoA reductase
(3-hydroxy-3-methyl-glutaryl-CoA reductase). Example statins
include atorvastatin (LIPITOR.RTM.), fluvastatin (LESCOL.RTM.),
lovastatin (MEVACOR.RTM., ALTOCOR.RTM., not marketed in the UK),
pravastatin (PRAVACHOL.RTM., SELEKTINE.RTM., LIPOSTAT.RTM.),
rosuvastatin (CRESTOR.RTM.), simvastatin (ZOCOR.RTM.). There are
two groups of statins: (1) Fermentation-derived: such as
lovastatin, simvastatin and pravastatin, and (2) Synthetic statins:
such as fluvastatin, atorvastatin, cerivastatin and
rosuvastatin.
[0127] Statins also have indirect effects on cholesterol metabolism
by upregulating hepatic expression of the LDL receptor and thus can
lower circulating LDL-C by as much as 50% and provide significant
protection against CVD (Baigent et al., Lancet 366: 1267-1278,
2005). There is also growing evidence that the statins provide
cardiovascular protection by mechanisms that are independent of
their LDL-C lowering effect, including anti-inflammatory and
anti-apoptotic activities and also by improving endothelial cell
function (Jain & Ridker, Nat Rev Drug Discov 4: 977-987, 2005).
The mechanisms, however, by which statins mediate these so called
pleiotropic effects on atherosclerosis are largely unknown.
[0128] Stroke (ischemic stroke): The rapidly developing loss of
brain function due to a disturbance in the blood supply to the
brain. There are two categories of stroke, "ischemic stroke" and
"hemorrhagic stroke." Ischemic stroke refers to a condition that
occurs when an artery to or in the brain is partially or completely
blocked such that the oxygen demand of the tissue exceeds the
oxygen supplied. Ischemic stroke is also referred to as "cerebral
ischemia." Deprived of oxygen and other nutrients following an
ischemic stroke, the brain suffers damage as a result of the
stroke. Ischemic stroke is by far the most common kind of stroke,
accounting for about 80% of all strokes.
[0129] Ischemic stroke can be caused by several different kinds of
diseases. The most common problem is narrowing of the arteries in
the neck or head. This is most often caused by atherosclerosis, or
gradual cholesterol deposition. If the arteries become too narrow,
blood cells may collect in them and form blood clots (thrombi).
These blood clots can block the artery where they are formed
(thrombosis), or can dislodge and become trapped in arteries closer
to the brain (embolism). Another cause of stroke is blood clots in
the heart, which can occur as a result of irregular heartbeat (for
example, atrial fibrillation), myocardial infarction, or
abnormalities of the heart valves, such as aortic valvular
insufficiency.
[0130] Therapeutically effective amount: A quantity of a specified
agent (or combination of agents) sufficient to achieve a desired
effect in a subject being treated with that agent.
[0131] Transformed: A transformed cell is a cell into which has
been introduced a nucleic acid molecule by molecular biology
techniques. As used herein, the term transformation encompasses all
techniques by which a nucleic acid molecule might be introduced
into such a cell, including transfection with viral vectors,
transformation with plasmid vectors, and introduction of naked DNA
by electroporation, lipofection, and particle gun acceleration.
[0132] Vasculopathy: A disease of the blood vessels. An
"age-related vasculopathy" is a disease of the blood vessels that
is associated with advanced age. One specific, non-limiting
vasculopathy is atherosclerosis. Other vasculopathies include, but
are not limited to, diabetic associated vasculopathy, hypertension
associated vasculopathy, Burger's disease associated vasculopathy
and scleroderma associated vasculopathy. It is understood that
"endothelial dysfunction" typically refers to an insufficiency in
the production or response to nitric oxide.
[0133] Vasoconstriction: The diminution of the caliber or
cross-sectional area of a blood vessel, for instance constriction
of arterioles leading to decreased blood flow to a body part. This
can be caused by a specific vasoconstrictor, an agent (for instance
a chemical or biochemical compound) that causes, directly or
indirectly, constriction of blood vessels. Such an agent can also
be referred to as a vasohypertonic agent, and is said to have
vasoconstrictive activity. A representative category of
vasoconstrictors is the vasopressor (from the term pressor, tending
to increase blood pressure), which term is generally used to refer
to an agent that stimulates contraction of the muscular tissue of
the capillaries and arteries.
[0134] Vasoconstriction also can be due to vasospasm, inadequate
vasodilatation, thickening of the vessel wall, or the accumulation
of flow-restricting materials on the internal wall surfaces or
within the wall itself. Vasoconstriction is a major presumptive or
proven factor in aging and in various clinical conditions including
progressive generalized atherogenesis, myocardial infarction,
stroke, hypertension, glaucoma, macular degeneration, migraine,
hypertension and diabetes mellitus, among others.
[0135] Vector: A nucleic acid molecule as introduced into a host
cell, thereby producing a transformed host cell. A vector may
include nucleic acid sequences that permit it to replicate in a
host cell, such as an origin of replication. A vector may also
include one or more selectable marker genes and other genetic
elements known in the art.
[0136] Vitamin E: Term that refers to a group of compounds that
include tocopherols and tocotrienols. The molecules that contribute
.alpha.-tocopherol activity in a vitamin E preparation are four
tocopherols and four tocotrienols, identified by the prefixes
alpha-(.alpha.-), beta- (.beta.-), gamma- (.gamma.-), and delta-
(.delta.-) Natural tocopherols occur in the RRR-configuration only;
synthetic forms contain eight different stereoisomers and may be
referred to as `all-rac`-.alpha.-tocopherol. Of the many different
forms of vitamin E, .gamma.-tocopherol a common form found in the
North American diet; .gamma.-tocopherol can be found in corn oil,
soybean oil, margarine, and dressings. .alpha.-tocopherol, the most
biologically active form of vitamin E, is the second-most common
form of vitamin E in the diet. This variant can be found most
abundantly in wheat germ oil, sunflower, and safflower oils.
Vitamin E preparations (either single compounds or mixtures) can
readily be obtained commercially.
[0137] 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 invention 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 invention, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references 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.
III. Overview of Several Embodiments
[0138] There is provided herein in a first embodiment a lipoprotein
targeting protease inhibitor peptide having the generic structure:
T-I, in which T is a hydrophobic entity comprising a vitamin E
(VitE), an acyl chain, or cholesterol; and I is a peptide-based
protease inhibitor; where T is covalently attached directly to I,
or indirectly by way of a hydrophilic linker L (the latter
resulting in the generic structure: T-L-I).
[0139] In example embodiments of the lipoprotein targeting protease
inhibitor peptide of, the hydrophobic lipoprotein targeting moiety
T is a vitamin E selected from the group consisting of:
alpha-tocopherol, beta-tocopherol, gamma-tocopherol,
delta-tocopherol, alpha-tocotrienol, beta-tocotrienol,
gamma-tocotrienol, or delta-tocotrienol. In additional example
embodiments, T is an acyl chain 4 to 24 carbons in length, with any
degree of hydrogen saturation.
[0140] The protease inhibitor component I can be any of myriad
peptide-based protease inhibitors (antiproteases), including
peptide-based inhibitors of elastase, matrix metalloprotease (MMP),
cathepsin, chymase, thrombin, coagulation factor IX, coagulation
factor X, urokinase-type plasminogen activator (uPA), tissue-type
plasminogen activator (tPA), and proteolytic components of the
complement cascade (C1r, C1s, MASPs 1-3, C2, Factor B, Factor D or
Factor I). By way of example, in certain embodiments of the
lipoprotein targeting protease inhibitor peptide, the peptide-based
inhibitor I: inhibits elastase, and comprises the sequence
Ala-Ala-Pro-Val (SEQ ID NO: 1); or inhibits elastase, and comprises
the general structure R.sub.1-W p-X
n-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-Y m-R.sub.2 (SEQ ID NO: 15),
in which AA.sub.1 is -Arg-, -Phg- and -Nle- or is a bond; AA.sub.2
is -Ala-, -Phg-, -Cit- and -Nle-; AA.sub.3 is -Trp-, -Val- and
-Tyr-; and AA.sub.4: -Phg- and -Gly-; or inhibits elastase, and
comprises a constrained or .beta.-hairpin peptide as shown in Tsai
et al., Table 2 or U.S. Pat. No. 8,658,604; or inhibits elastase,
and comprises Pep4 (KRCCPDTCGIKCL; positions 3-16 pf SEQ ID NO: 8)
or Pep4M (KRMMPDTMGIKML; positions 3-16 of SEQ ID NO: 9); or
inhibits matrix metalloprotease, and comprises the sequence of an
inhibitory peptide in Ndinguri et al., Molecules 17:14230-14248,
2012; or inhibits cathepsin, and comprises the structure
Z-Phe-Gly-NHO-Bz, in which Z=carboxybenzyl and Bz=benzyl; or
inhibits cathepsin, and comprises the structure Z-Phe-Phe-DK or
Z-Phe-Phe-CHN.sub.2; or inhibits chymase, and comprises the
structure Z-Arg-Glu-Thr-Phep(OPh).sub.2; or inhibits thrombin
and/or coagulation factors IX and X, and is selected from Hirudin
(SEQ ID NO: 11) or a derivative thereof, such as Lepirudin or
Desirudin; or inhibits plasminogen activator, and comprises
aprotinin (SEQ ID NO: 12) or the plasminogen activator inhibitor
type 1 (PAI-1)-derived peptide EEIIMD (positions 3-8 of SEQ ID NO:
10). In yet another embodiment, the peptide-based inhibitor
inhibits elastase and comprises Ala-Ala-Pro-Val-chloromethylketone
(AAPV-CMK) (SEQ ID NO: 3).
[0141] Optionally, the lipoprotein targeting protease inhibitor
peptide may comprise a linker L, for instance which is a
hydrophilic linker comprising polyethylene glycol (PEG) (of any
length, for instance of MW 1000, 2000, 2500, 5000, and so forth) or
succinimide.
[0142] Specifically contemplated lipoprotein targeting protease
inhibitor peptides comprising the structure: VitE-AAPV (SEQ ID NO:
4); VitE-PEG-AAPV (SEQ ID NO: 5); VitE-PEG-AAPV-CMK (SEQ ID NO: 6);
VitE-AAPV-CMK (SEQ ID NO: 7); VitE-PEG-KRCCPDTCGIKCL (SEQ ID NO:
8); VitE-PEG-KRMMPDTMGIKML (SEQ ID NO: 9); VitE-PEG-EEIIMD (SEQ ID
NO: 10); VitE-PEG-hirudin; VitE-PEG-lepirudin; VitE-PEG-desirudin;
SEQ ID NO: 13; or SEQ ID NO: 14.
[0143] Also provided herein are pharmaceutical compositions,
comprising at least one lipoprotein targeting protease inhibitor
peptide, and a pharmaceutically acceptable carrier. Optionally, the
peptide in such a composition is contained in or part of a
lipoprotein, such as a HDL.
[0144] Methods comprising administering such a pharmaceutical
composition to a subject are also provided. By way of example, such
a method may be a method of treating a protease-mediated disease or
defect in the subject. In embodiments of these methods, the I
component in the lipoprotein targeting protease inhibitor peptide
is selected to complement/treat the protease-mediated disease or
defect of the subject.
[0145] Also provided are methods of producing HDL with enriched
protease inhibitor (antiprotease) activity, comprising contacting
HDL with a lipoprotein targeting protease inhibitor peptide as
provided herein. In embodiments of such methods, the HDL is
reconstituted HDL (rHDL) and the method is carried out ex vivo. In
other embodiments of such methods, contacting HDL with the
lipoprotein targeting protease inhibitor peptide occurs in the
bloodstream of a subject. Also provided herein are protease
inhibitor enriched HDL produced by these methods.
[0146] Yet additional embodiments are protease inhibitor enriched
HDL, comprising HDL and a lipoprotein targeting protease inhibitor
peptide as described herein. In examples of this embodiment, the
protease inhibitor enriched HDL reconstituted HDL (rHDL).
IV. Passenger Protein-Enriched HDL and Uses Thereof
[0147] It has been shown that HDL particles can be "loaded" with
therapeutic proteins, including native anti-proteases and
antioxidants. Such therapeutic-protein enriched HDLs are proposed
for use in various therapeutic contexts. See, for instance,
International Application Publication No. WO2011006994 A1, entitled
"HDL COMPRISING A THERAPEUTIC AGENT AND USE IN THERAPY", which
teaches methods to make and use HDL that comprise an agent such as
an antiprotease, antioxidant, anti-mitotic, iron metabolism agent,
or anti-apoptotic agent, for use as a medicament. The teachings of
that publication are hereby incorporated herein by reference in
their entirety.
[0148] Conditions treatable with the A1AT-enriched HDL particles
include, but are not limited to, hyperlipidemia (e.g.,
hypercholesterolemia), cardiovascular disease (e.g.,
atherosclerosis), restenosis (e.g., atherosclerotic plaques),
peripheral vascular disease, acute coronary syndrome, reperfusion
myocardial injury, asthma, A1AT deficiency, chronic pulmonary
obstructive disorder and the like.
V. Lipoprotein-Targeting Protease Inhibitor Peptides (fusions) and
Uses Thereof
[0149] Described herein is the discovery and development of
lipoprotein-targeting protease inhibitor peptides, for instance
which can target protease inhibitor peptides (rather than native,
full-length protease proteins) to lipoproteins, including
specifically HDL particles.
[0150] The prototypical lipoprotein-targeting protease inhibitor
fusion protein includes a small peptide inhibitor of elastase (for
instance, a small peptide derived from A1AT) attached via a
hydrophillic linker molecule to a hydrophobic "targeting" moiety
(having affinity for a lipoprotein, such as HDL) such as vitamin E,
an acyl chain, or cholesterol or the like. The lipoprotein
targeting protease inhibitor peptides provided herein present
several significant advances upon the prior existing methods for
treating A1AT deficiency. Full length A1AT protein is replaced with
a known small peptide inhibitor of elastase (the natural target
protease of A1AT), a small tetra-peptide with the sequence
Ala-Ala-Pro-Val-chloromethyl ketone (CMK) (SEQ ID NO: 3). This
peptide is conjugated to a lipoprotein targeting motif using amine
reactive chemistry. By way of example, the peptide has been linked
to a Vitamin E molecule with a polyethylene glycol spacer arm to
distance the functional AAPV peptide from the targeting moiety and
to provide improved solubility. This approach is expected to
provide improved efficacy over the current standard of care (A1AT
infusion) because, for instance, the binding of A1AT to HDL has
been shown to provide greatly improved efficacy in animal models of
COPD (Meilhac et al., Handb Exp Pharmacol. 224:509-526, 2015). This
is because HDL facilitates the movement of A1AT to sites of
inflammation. This tissue targeting effect is expected to be
recapitulated with the lipid-targeted fusion peptide structures
described herein. Additionally, the resultant peptide is a small
molecule of about 2.5 kDa, much smaller than the full length A1AT
protein (52 kDa). An HDL particle can generally accommodate only
one molecule of A1AT, whereas many copies of our VitE-PEG-AAPV
peptide can reside on an HDL particle providing a significant
increase in potency. Similar benefits are realized with additional
embodiments of the provided lipoprotein-targeted antiprotease
molecules, using alternative antiprotease peptides such as serpins
and fragments thereof. Thus, the prototypical targeted antiprotease
peptide can be modified to inhibit almost any protease by modifying
its protease inhibitor moiety.
[0151] With the provision herein of a prototype
lipoprotein-targeting protease inhibitor fusion peptide, there is
now enabled an entire genus of such peptides. Thus, there are
provided herein lipoprotein targeting protease inhibitors with the
following generalized design:
T-I
in which T (the targeting domain) is a hydrophobic component, such
as Vitamin E (alpha, beta, gamma, or delta tocopherol or
tocotrienol), an acyl chain (a fatty acid ranging in chain length
from 4 to 24 carbon atoms with any degree of hydrogen saturation),
or cholesterol; component I is a peptide inhibitor of a protease
(an antiprotease peptide); and optionally included between T and I
is a linker L (thus, T-L-I), which when present is a hydrophilic
linker, for instance comprising or consisting of polyethylene
glycol (PEG) or succinimide, which links the targeting (T) domain
to the inhibitor (I) domain. Particularly contemplated are example
constructed peptides in which the component I is a peptide derived
inhibitor of Elastase, Matrix Metalloprotease, Cathepsin, Chymase,
Thrombin, coagulation factors IX and X, Plasminogen activators,
urokinase-type (uPA) and the tissue-type (tPA), or a proteolytic
component of the complement cascade (C1r, C1s, MASPs 1-3, C2 and
Factor B, Factor D and Factor I).
[0152] In some embodiments, the component I is a peptide inhibitor
of (leukocyte) elastase; such peptide inhibitors are known in the
art. These include peptide chloromethyl ketones described by Tuhy
& Powers (FEBS Lett 50:359-362, 1975), including linear
peptides containing electrophilic groups such as
Ala-Ala-Pro-Val-Chloromethylketone (CMK) (SEQ ID NO: 3; available
commercially for instance from MP Biomedicals, Santa Ana, Calif.).
Also contemplated are peptides described in International Patent
Publication WO/2010091893 ("Peptides Used in the Treatment and/or
Care of the Skin, Mucous Membranes and/or Scalp and Their Use in
Cosmetic or Pharmaceutical Compositions"), including those defined
with the general formula (I):
[0153]
R.sub.1-W.sub.p-X.sub.n-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-Y.sub.m-
-R.sub.2 (SEQ ID NO: 15) in which AA.sub.1: -Arg-, -Phg- and -Nle-
or is a bond; AA.sub.2: -Ala-, -Phg-, -Cit- and -Nle-; AA.sub.3:
-Trp-, -Val- and -Tyr-; AA.sub.4: -Phg- and -Gly-; W, X and Y are
independently selected from the group consisting of coded or
uncoded amino acids; p, n and m range between 0 and 1; R.sub.1 is
selected from the group consisting of H, substituted or
unsubstituted non-cyclic aliphatic group, substituted or
unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted aryl, substituted or unsubstituted aralkyl and
R.sub.5--CO--; R.sub.2 is selected from the group consisting of
--NR.sub.3R4, --OR3 and --SR.sub.3; wherein R.sub.3 and R.sub.4 are
independently selected from the group consisting of H, substituted
or unsubstituted non-cyclic aliphatic group, substituted or
unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl,
substituted or unsubstituted heteroarylalkyl, substituted or
unsubstituted aryl and substituted or unsubstituted aralkyl;
wherein R.sub.5 is selected from the group consisting of H,
substituted or unsubstituted non-cyclic aliphatic group,
substituted or unsubstituted alicyclyl, substituted or
unsubstituted aryl, substituted or unsubstituted aralkyl,
substituted or unsubstituted heterocyclyl and substituted or
unsubstituted heteroarylalkyl; and provided that when AA.sub.1 is a
bond, AA.sub.2 is -Phg- and AA.sub.3 is -Trp-. R.sub.1 and R.sub.2
groups are bound to the amino-terminal (N-terminal) and
carboxy-terminal (C-terminal) of the peptide sequences
respectively. Additional appropriate peptide inhibitors of elastase
are described in Tsai & Hwang (Expert Opin Ther Patents
25:1145-1158, 2015), including the constrained or .beta.-hairpin
peptides provided in Table 2 of that paper (see also U.S. Pat. No.
8,658,604, "Template-Fixed Beta-Hairpin Peptidomimetics with
Protease Inhibitory Activity").
[0154] In additional embodiments, the component I is a peptide
inhibitor of matrix metalloprotease, such as those described by
Ndinguri et al. (Molecules 17:14230-14248, 2012).
[0155] In additional embodiments, the component I is a peptide
inhibitor of cathepsin. One example of such an inhibitor is
Z-Phe-Gly-NHO-Bz (available commercially from Santa Cruz
Biotechnology, for instance); another is Z-Phe-Phe-DK (available
commercially from MP Biomedical). See also Schaschke et al. (Biol.
Chem 383:849-852, 2002), describing epoxysuccinyl peptide-derived
cathepsin B inhibitors.
[0156] In additional embodiments, the component I is a peptide
inhibitor of plasmin, such as those described in Ding et al.
(Toxicon 106:7-13, 2015).
[0157] In additional embodiments, component I is a peptide
inhibitor of Chymase. On such inhibitor is
Z-Arg-Glu-Thr-Phep(OPh).sub.2 (available commercially from MP
Biomedical).
[0158] In additional embodiments, component I is a peptide
inhibitor of Thrombin and/or coagulation factors IX and X. Such
peptide inhibitors include hirudin (SEQ ID NO: 11) and derived
peptides such as lepirudin and desirudin; see, for instance, Lee et
al. (Br J Clin Pharmacol 72:581-592, 2011); Greinacher &
Warkentin (Thromb Haemost 99:819-829, 2008).
[0159] In yet additional embodiments, component I is a peptide
inhibitor of a Plasminogen activators, such as urokinase-type (uPA)
and/or tissue-type (tPA). These include for instance Aprotinin
(available commercially from Sigma-Aldrich), and the PAI-1-derived
peptide EEIIMD (positions 3-8 of SEQ ID NO: 10) (Armstead et al.,
Am J Physiol Heart Circ Physiol 299:H76-H80, 2010)>
[0160] In additional embodiments, the component I is a peptide
inhibitor of a proteolytic component of the complement cascade
(C1r, C1s, MASPs 1-3, C2 and Factor B, Factor D and Factor I).
[0161] Specifically contemplated are lipoprotein targeting protease
inhibitor peptides with the following components/structure:
VitE-AAPV (SEQ ID NO: 4); VitE-PEG-AAPV (SEQ ID NO: 5);
VitE-PEG-AAPV-CMK (SEQ ID NO: 6); VitE-AAPV-CMK (SEQ ID NO: 7);
VitE-PEG-KRCCPDTCGIKCL (SEQ ID NO: 8); VitE-PEG-KRMMPDTMGIKML (SEQ
ID NO: 9); VitE-PEG-EEIIMD (SEQ ID NO: 10); VitE-PEG-hirudin;
VitE-PEG-lepirudin; VitE-PEG-desirudin; SEQ ID NO: 13; or SEQ ID
NO: 14.
[0162] Conditions treatable with A1AT-peptide (and other elastase
inhibiting peptide) containing lipoprotein targeted antiprotease
molecules and particles described herein include, but are not
limited to, hyperlipidemia (e.g., hypercholesterolemia),
cardiovascular disease (e.g., atherosclerosis), restenosis (e.g.,
atherosclerotic plaques), peripheral vascular disease, acute
coronary syndrome, reperfusion myocardial injury, asthma, A1AT
deficiency, chronic pulmonary obstructive disorder (COPD),
emphysema, and the like. It is believed that HDL particles that are
enriched with the herein described A1AT-peptide fusion will be
directed for tissue uptake to sites of inflammation, including for
instance the lung (Meilhac et al., Handb Exp Pharmacol.
224:509-526, 2015) (particularly useful in treating A1AT deficiency
and COPD/Emphysema for instance), as well as vessel walls (Von
Eckardstein et al., Curr Opin Lipidol. 27(3):264-273, 2016; Hazen
et al., Arterioscler Thromb Vasc Biol. 30(2):138, 2010. doi:
10.1161/ATVBAHA.109.201897) (particularly useful for treating
atherosclerosis, and unstable plaque).
[0163] The provided lipoprotein targeted antiprotease peptides can
also be used in the treatment of other protease-mediated disease,
including for instance HIV (Hazen et al., Antimicrob Agents
Chemother. 51(9):3147-3154, 2007), hepatitis, and cancer. For
instance, it is recognized that antiproteases are useful in the
treatment of cancer. (Li et al., Cancer Res. 64(23):8657-65, 2004;
Uetsuji, Surg Today; 22(5):439-442, 1992). It has also been
reported that elastase is able to degrade intracellular substrates
leading to increased tumor growth (Houghton et al., Nat Med.
16(2):219-223, 2010).
[0164] See also International Application Publication No.
WO2011006994 A1 (INSERM, "HDL COMPRISING A THERAPEUTIC AGENT AND
USE IN THERAPY"); Ortiz-Munoz et al., FASEB J 23:3129-3139, 2009;
Tran-Dinh et al., Br J Pharmacol 169:493-511, 2013; Lapergue et
al., Stroke 41:1536-1542, 2010; Mutharasan et al., J Mater Chem B
4:188-197, 2016;
[0165] Further, it is recognized that the lipoprotein targeted
antiprotease peptide molecules described herein have utility in
research, for instance to characterize the structure and/or
function of lipoproteins as well as the peptide molecules and their
component parts.
[0166] Construction or assembly of lipoprotein targeted
antiprotease peptide molecules described herein can be carried out
using readily available chemical synthesis processes and isolation
techniques, as will be recognized by one of skill in the art. As
demonstrated herein, commercially prepared and activated "T-L"
conjugates (e.g. vitamin E-PEG-NHS) can be utilized in making
fusion peptides of the discloser. In such examples, synthesis of
the final desired "T-L-I" structure involves conjugation of the TL
and I via NHS chemistry.
[0167] Methods for synthesis of cholesterol-derivatized (and other
lipid-derivatized) peptides are known in the art. The cholesterol
(or other lipid) moiety is generally attached to the peptide via a
thioether linkage with the thiol group of an extra cysteine
residue, added C-terminally to the select (protease inhibitory)
sequence. A thioether bond is generally used as an attachment
point, since it provides both for non hydrolyzable anchoring to the
membrane, and for an easy preparation of the vaccine via
chemoselective methods. Chemoselective reaction between bromoacetyl
groups and free thiols are described, for instance, in Zeng et al.,
Vaccine 19, 3843-3852, 2001 (incorporated herein by reference).
##STR00001##
[0168] Bromoacetyl compounds can be made using commercially
available compounds or by well-known methods from commercially
available compounds. For instance, a method for synthesizing
Bromoacetyl-cholesterol (below) is provided in international
Application Publication No. WO2009053339 A2, which is incorporated
herein by reference.
##STR00002##
[0169] Similar techniques can be used to produce other
bromoacetyl-lipids for use in fusion molecules as described
herein.
[0170] As illustrated in the Examples with vitamin E-PEG-NHS
starting materials, cholesterol is also commercially available as
cholesterol-PEG-NHS for use in amine reactive chemistry, for
instance from Nanocs Inc. (NY) and other commercial sources.
[0171] Optionally, additional embodiments of a linker for use with
the disclosed conjugate peptides is a heterobifunctional
polyalkyleneglycol linker having the general structure shown
below:
##STR00003##
wherein A and B include different reactive groups, x is an integer
from 2 to 10 (such as 2, 3 or 4), and y is an integer from 1 to 50,
for example, from 2 to 30 such as from 3 to 20 or from 4 to 12. One
or more hydrogen atoms can be substituted for additional functional
groups such as hydroxyl groups, alkoxy groups (such as methoxy and
ethoxy), halogen atoms (F, Cl, Br, I), sulfato groups and amino
groups (including mono- and di-substituted amino groups such as
dialkyl amino groups.
[0172] A and B of the linker can independently include a
carbonyl-reactive group, an amine-reactive group, a thiol-reactive
group or a photo-reactive group, but are not the same. Examples of
carbonyl-reactive groups include aldehyde- and ketone-reactive
groups like hydrazine derivatives and amines. Examples of
amine-reactive groups include active esters such as NHS or
sulfo-NHS, isothiocyanates, isocyanates, acyl azides, sulfonyl
chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates,
aryl halides, imidoesters, anhydrides and the like. Examples of
thiol-reactive groups include non-polymerizable Michael acceptors,
haloacetyl groups (such as iodoacetyl), alkyl halides, maleimides,
aziridines, acryloyl groups, vinyl sulfones, benzoquinones,
aromatic groups that can undergo nucleophilic substitution such as
fluorobenzene groups (such as tetra and pentafluorobenzene groups),
and disulfide groups such as pyridyl disulfide groups and thiols
activated with Ellman's reagent. Examples of photo-reactive groups
include aryl azide and halogenated aryl azides. Alternatively, A
and/or B can be a functional group that reacts with a specific type
of reactive group. For example, A and/or B can be an amine group, a
thiol group, or a carbonyl-containing group that will react with a
corresponding reactive group (such as an amine-reactive group,
thiol-reactive group or carbonyl-reactive group, respectively) that
has been introduced or is otherwise present on a hapten and/or a
tyramine or tyramine derivative. Additional examples of each of
these types of groups will be apparent to those skilled in the art.
Further examples and information regarding reaction conditions and
methods for exchanging one type of reactive group for another are
provided in Hermanson, "Bioconjugate Techniques," Academic Press,
San Diego, 1996, which is incorporated by reference herein.
VI. Synthesis and Purification of the Peptide Domain
[0173] The peptide/protein-derived protease inhibitor domain of the
lipoprotein-targeted inhibitor peptides of the disclosure can be
prepared using virtually any technique known to one of ordinary
skill in the art for the preparation of peptides. For example, the
peptides can be prepared using step-wise solution or solid phase
peptide syntheses, or recombinant DNA techniques, or the
equivalents thereof.
[0174] A. Chemical Synthesis
[0175] Peptides for use in the fusion molecules provided herein
comprised of amino acids of either the D- or L-configuration can be
readily synthesized by automated solid phase procedures well known
in the art. Suitable syntheses can be performed by utilizing
"T-boc" or "F-moc" procedures. Techniques and procedures for solid
phase synthesis are described in Solid Phase Peptide Synthesis: A
Practical Approach, by E. Atherton and R. C. Sheppard, published by
IRL, Oxford University Press, 1989. Alternatively, the multi-domain
peptides may be prepared by way of segment condensation, as
described, for example, in Liu et al., Tetrahedron Lett.
37:933-936, 1996; Baca et al., J. Am. Chem. Soc. 117:1881-1887,
1995; Tam et al., Int. J. Peptide Protein Res. 45:209-216, 1995;
Schnolzer and Kent, Science 256:221-225, 1992; Liu and Tam, J. Am.
Chem. Soc. 116:4149-4153, 1994; Liu and Tam, Proc. Natl. Acad. Sci.
USA 91:6584-6588, 1994; and Yamashiro and Li, Int. J. Peptide
Protein Res. 31:322-334, 1988). This is particularly the case with
glycine containing peptides. Other methods useful for synthesizing
the multi-domain peptides of the disclosure are described in
Nakagawa et al., J. Am. Chem. Soc. 107:7087-7092, 1985.
[0176] Additional exemplary techniques known to those of ordinary
skill in the art of peptide and peptide analog synthesis are taught
by Bodanszky, M. and Bodanszky, A., The Practice of Peptide
Synthesis, Springer Verlag, New York, 1994; and by Jones, J., Amino
Acid and Peptide Synthesis, 2nd ed., Oxford University Press, 2002.
The Bodanszky and Jones references detail the parameters and
techniques for activating and coupling amino acids and amino acid
derivatives. Moreover, the references teach how to select, use and
remove various useful functional and protecting groups.
[0177] Peptides of the disclosure comprised of amino acids of
either the D- or L-configuration can also be readily purchased from
commercial suppliers of synthetic peptides. Such suppliers include,
for example, Advanced ChemTech (Louisville, Ky.), Applied
Biosystems (Foster City, Calif.), Anaspec (San Jose, Calif.), and
Cell Essentials (Boston, Mass.). Specific inhibitor peptides are
also available from other sources, as will be recognized by those
of ordinary skill in the art.
[0178] B. Recombinant Synthesis
[0179] If the peptide is composed entirely of gene-encoded amino
acids, or a portion of it is so composed, the multi-domain peptide
or the relevant portion can also be synthesized using conventional
recombinant genetic engineering techniques. For recombinant
production, a polynucleotide sequence encoding the multi-domain
peptide is inserted into an appropriate expression vehicle, that
is, a vector which contains the necessary elements for the
transcription and translation of the inserted coding sequence, or
in the case of an RNA viral vector, the necessary elements for
replication and translation. The expression vehicle is then
transfected into a suitable target cell which will express the
multi-domain peptide. Depending on the expression system used, the
expressed peptide is then isolated by procedures well-established
in the art. Methods for recombinant protein and peptide production
are well known in the art (see, e.g., Sambrook et al. (ed.),
Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., vol. 1-3,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989, Ch. 17 and Ausubel et al. Short Protocols in Molecular
Biology, 4.sup.th ed., John Wiley & Sons, Inc., 1999).
[0180] To increase efficiency of production, the polynucleotide can
be designed to encode multiple units of the multi-domain peptide
separated by enzymatic cleavage sites. The resulting polypeptide
can be cleaved (e.g., by treatment with the appropriate enzyme) in
order to recover the peptide units. This can increase the yield of
peptides driven by a single promoter. In one embodiment, a
polycistronic polynucleotide can be designed so that a single mRNA
is transcribed which encodes multiple peptides, each coding region
operatively linked to a cap-independent translation control
sequence, for example, an internal ribosome entry site (IRES). When
used in appropriate viral expression systems, the translation of
each peptide encoded by the mRNA is directed internally in the
transcript, for example, by the IRES. Thus, the polycistronic
construct directs the transcription of a single, large
polycistronic mRNA which, in turn, directs the translation of
multiple, individual peptides. This approach eliminates the
production and enzymatic processing of polyproteins and can
significantly increase yield of peptide driven by a single
promoter.
[0181] A variety of host-expression vector systems may be utilized
to express the peptides described herein. These include, but are
not limited to, microorganisms such as bacteria transformed with
recombinant bacteriophage DNA or plasmid DNA expression vectors
containing an appropriate coding sequence; yeast or filamentous
fungi transformed with recombinant yeast or fungi expression
vectors containing an appropriate coding sequence; insect cell
systems infected with recombinant virus expression vectors (e.g.,
baculovirus) containing an appropriate coding sequence; plant cell
systems infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or
transformed with recombinant plasmid expression vectors (e.g., Ti
plasmid) containing an appropriate coding sequence; or animal cell
systems.
[0182] The expression elements of the expression systems vary in
their strength and specificities. Depending on the host/vector
system utilized, any of a number of suitable transcription and
translation elements, including constitutive and inducible
promoters, can be used in the expression vector. For example, when
cloning in bacterial systems, inducible promoters such as pL of
bacteriophage .lamda., plac, ptrp, ptac (ptrp-lac hybrid promoter)
and the like can be used. When cloning in insect cell systems,
promoters such as the baculovirus polyhedron promoter can be used.
When cloning in mammalian cell systems, promoters derived from the
genome of mammalian cells (e.g., metallothionein promoter) or from
mammalian viruses (e.g., the adenovirus late promoter, the vaccinia
virus 7.5 K promoter) can be used.
[0183] C. Purification
[0184] The peptides of the disclosure can be purified by many
techniques well known in the art, such as reverse phase
chromatography, high performance liquid chromatography, ion
exchange chromatography, size exclusion chromatography, affinity
chromatography, gel electrophoresis, and the like. The actual
conditions used to purify a particular multi-domain peptide or
peptide analog will depend, in part, on synthesis strategy and on
factors such as net charge, hydrophobicity, hydrophilicity, and the
like, and will be apparent to those of ordinary skill in the
art.
[0185] For affinity chromatography purification, any antibody which
specifically binds the multi-domain peptide or peptide analog may
be used. For the production of antibodies, various host animals,
including but not limited to, rabbits, mice, rats, and the like,
may be immunized by injection with a multi-domain peptide or
peptide analog. The multi-domain peptide or peptide analog can be
attached to a suitable carrier (e.g., BSA) by means of a side chain
functional group or linker attached to a side chain functional
group. Various adjuvants may be used to increase the immunological
response, depending on the host species, including but not limited
to, Freund's (complete and incomplete), mineral gels (e.g.,
aluminum hydroxide), surface active substances (e.g., lysolecithin,
pluronic polyols, polyanions, and oil emulsions), keyhole limpet
hemocyanin, dinitrophenol, and potentially useful human adjuvants
such as BCG (bacilli Calmette-Guerin) and Corynebacterium
parvum.
[0186] Booster injections can be given at regular intervals, and
antiserum harvested when the antibody titer thereof, as determined
semi-quantitatively, for example, by double immunodiffusion in agar
against known concentrations of the antigen, begins to fall. See,
e.g., Ouchterlony et al., Handbook of Experimental Immunology,
Wier, D. (ed.), Chapter 19, Blackwell, 1973. A plateau
concentration of antibody is usually in the range of 0.1 to 0.2
mg/ml of serum (about 12 .mu.M). Affinity of the antisera for the
antigen is determined by preparing competitive binding curves, as
described, for example, by Fisher (Manual of Clinical Immunology,
Ch. 42, 1980).
[0187] Monoclonal antibodies to a peptide (either a domain to be
used in a lipoprotein-targeting protease inhibitor fusion peptide,
or the fusion peptide molecule itself) may be prepared using any
technique which provides for the production of antibody molecules
by continuous cell lines in culture, for example the classic method
of Kohler & Milstein (Nature 256:495-97, 1975), or a derivative
method thereof. Briefly, a mouse is repetitively inoculated with a
few micrograms of the selected protein immunogen (e.g., a
multi-domain peptide or peptide analog) over a period of a few
weeks. The mouse is then sacrificed, and the antibody-producing
cells of the spleen isolated. The spleen cells are fused by means
of polyethylene glycol with mouse myeloma cells, and the excess
unfused cells destroyed by growth of the system on selective media
comprising aminopterin (HAT media). The successfully fused cells
are diluted and aliquots of the dilution placed in wells of a
microtiter plate where growth of the culture is continued.
Antibody-producing clones are identified by detection of antibody
in the supernatant fluid of the wells by immunoassay procedures,
such as enzyme-linked immunosorbent assay (ELISA), as originally
described by Engvall (Meth. Enzymol., 70:419-39, 1980), or a
derivative method thereof. Selected positive clones can be expanded
and their monoclonal antibody product harvested for use. Detailed
procedures for monoclonal antibody production are described in
Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New
York, 1999. Polyclonal antiserum containing antibodies can be
prepared by immunizing suitable animals with a polypeptide
comprising at least one multi-domain peptide or peptide analog,
which can be unmodified or modified, to enhance immunogenicity.
[0188] Antibody fragments may be used in place of whole antibodies
and may be readily expressed in prokaryotic host cells. Methods of
making and using immunologically effective portions of monoclonal
antibodies, also referred to as "antibody fragments," are well
known and include those described in Better & Horowitz, Methods
Enzymol. 178:476-96, 1989; Glockshuber et al., Biochemistry
29:1362-67, 1990; and U.S. Pat. No. 5,648,237 (Expression of
Functional Antibody Fragments); U.S. Pat. No. 4,946,778 (Single
Polypeptide Chain Binding Molecules); and U.S. Pat. No. 5,455,030
(Immunotherapy Using Single Chain Polypeptide Binding Molecules),
and references cited therein. Conditions whereby a
polypeptide/binding agent complex can form, as well as assays for
the detection of the formation of a polypeptide/binding agent
complex and quantitation of binding affinities of the binding agent
and polypeptide, are standard in the art. Such assays can include,
but are not limited to, Western blotting, immunoprecipitation,
immunofluorescence, immunocytochemistry, immunohistochemistry,
fluorescence activated cell sorting (FACS), fluorescence in situ
hybridization (FISH), immunomagnetic assays, ELISA, ELISPOT
(Coligan et al., Current Protocols in Immunology, Wiley, N Y,
1995), agglutination assays, flocculation assays, cell panning,
etc., as are well known to one of skill in the art.
[0189] Optionally, peptides and fusion molecules produced as
described herein may also be purified using HPLC or other chemical
laboratory means.
VII. Activity of Lipoprotein Targeting Protease Inhibitor
Peptides
[0190] Once a lipoprotein targeting protease inhibitor peptide (a
lipoprotein-mediated antiprotease) is produced, including variants
beyond the specific examples provided herein, the function(s) of
the peptide can be assayed using methods such as those described
herein, or methods readily recognized by those of skill in the
relevant art. Representative methods for testing activity are also
provided herein, including peptide affinity for and
binding/complexing with lipoprotein as well as protease
activity.
VIII. Incorporation of Lipoprotein Targeting Protease Inhibitor
Peptides into Lipoprotein Particles
[0191] It is contemplated that the lipoprotein targeting protease
inhibitor peptides described herein can be loaded/assembled into
lipoprotein particles, such as HDL particles, and the resultant
enriched particles used as therapeutic agents. The
production/assemble of such loaded particles is similar to the type
of assembly that could be used in the preparation of any
functionalized rHDL or nHDL. Representative methods are described
or incorporated herein
[0192] In general, HDL particles for use in such embodiments can be
synthetic preparations such as those described herein composed of
phospholipid and human purified or recombinant apoA-I (for
instance, assembled by reconstitution of dried lipids using a
sterile saline solution containing apoA-I); alternatively, they can
be native HDL particles isolated for instance from healthy
individuals.
[0193] By way of example, lipoprotein particles, such as HDL, may
be loaded (complexed, enriched) with a lipoprotein targeted
antiprotease as follows: incubate HDL (either reconstituted or
native) with the selected peptide agent at an appropriate
concentration under gentle agitation at 37.degree. C. for an
appropriate time, the concentration and time depending on the
affinity of the selected agent (and specifically, the lipoprotein
targeting component T of that agent) for the HDL; separate the
resultant mixture using potassium bromide gradient
ultracentrifugation; and collection and purification of the
enriched HDL, for instance using dialysis against a saline solution
or filtered using a centrifugal device.
[0194] Alternatively, HDL may be loaded by incubating with the
selected peptide agent under gentle agitation at 37.degree. C. for
an appropriate time; followed by filtration using a cut-off
centrifugal device. Free agent (not associated with the HLD)
partitions into the flow-through whereas enriched HDL remain in the
upper compartment.
[0195] A person having ordinary skill in the art will be aware of
the conditions for carrying out lipoprotein particle (e.g., HDL)
loading. For example, if the agent has a low affinity with the HDL,
the HDL will be incubated with a higher concentration of said agent
and for a longer time, than if the agent had a natural and high
affinity for the HDL. Likewise, a person of ordinary skill in the
art is able to select the appropriate molecular weight cutoff of
the centrifugal device for carrying out the required
filtrations.
IX. Incorporation of Lipoprotein Targeting Protease Inhibitor
Peptide(s) or Lipoproteins Containing Such Peptides into
Pharmaceutical Compositions
[0196] Pharmaceutical compositions that comprise at least one
lipoprotein targeting protease inhibitor peptide, or a lipoprotein
particle containing such a peptide, as described herein as an
active ingredient will normally be formulated with a solid, gel
(e.g., Matrigel), or liquid carrier, depending upon the particular
mode of administration chosen. The pharmaceutically acceptable
carriers and excipients useful in this invention are conventional.
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. Excipients
that can be included are, for instance, other proteins, such as
human serum albumin or plasma preparations. If desired, the
pharmaceutical composition to be administered may also contain
minor amounts of non-toxic auxiliary substances, such as wetting or
emulsifying agents, preservatives, and pH buffering agents and the
like, for example sodium acetate or sorbitan monolaurate.
[0197] The dosage form of the pharmaceutical composition will be
determined by the mode of administration chosen. For instance, in
addition to injectable fluids, topical and oral formulations can be
employed. Oral formulations will usually be solid (e.g., powders,
pills, tablets, or capsules, including for instance enteric coated
solid delivery forms), and preferentially will comprise peptides
rather than peptide-loaded lipoprotein formulations. 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.
[0198] The pharmaceutical compositions that comprise lipoprotein
targeting protease inhibitor peptide(s), or lipoprotein particles
containing such a peptide (e.g., a protease inhibitor enriched
lipoprotein particle), may be formulated in unit dosage form,
suitable for individual administration of precise dosages. One
possible unit dosage contains approximately 50 .mu.g of peptide or
protease inhibitor enhanced lipoprotein; alternatively, a unit dose
may contain 60 .mu.g, 70 .mu.g, 80 .mu.g, 90 .mu.g, 100 .mu.g or
more. The amount of active compound 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 an amount effective to achieve the desired effect in the subject
being treated. However, dosages ranges comparable to those
currently used in lipid-free A1AT replacement therapies (for
instance, 60 mg/kg weekly) may provide guidance from which ranges
could be optimized.
[0199] To extend the time during which the peptide is available in
the subject's system, the peptide can be provided as an implant, an
oily injection, or as a particulate system. The particulate system
can be a microparticle, a microcapsule, a microsphere, a
nanocapsule, or similar particle (Banga, "Parenteral Controlled
Delivery of Therapeutic Peptides and Proteins," in Therapeutic
Peptides and Proteins, Technomic Publishing Co., Inc., Lancaster,
Pa., 1995).
[0200] In some examples, the provided peptides are combined with a
pharmaceutically acceptable carrier (e.g., a phospholipid or other
type of lipid) or vehicle for administration to human or animal
subjects. In some embodiments, more than one disclosed peptide can
be combined to form a single preparation.
[0201] Additionally, in some embodiments the therapeutic agents may
be incorporated in implantable devices, such as vascular stents
placed directly in diseased blood vessels in the coronary, cerebral
or peripheral circulation, for instance to provide slow release of
the compound, thereby providing regional sustained release of the
therapeutic agents.
[0202] For subjects with peripheral artery disease (and other
systemic and arterial diseases), administration is, for example, by
intra-arterial (particularly intracoronary), or intrapericardial
injection. In some embodiments, the therapeutic agent is
administered systemically, such as by intravenous injection.
Additionally, in some embodiments the therapeutic agents may be
incorporated into or on an implantable device, such as vascular
stents placed directly in diseased blood vessels in the coronary or
cerebral circulation, and undergo slow release providing regional
sustained release of the therapeutic agents. Efficacy of treatment
is demonstrated, for example, by a regression of symptoms, for
example chest pressure or pain.
[0203] In another embodiment, it may be desirable to administer the
pharmaceutical compositions locally to the area in need of
treatment. This may be achieved by, for example, and not by way of
limitation, local or regional infusion or perfusion during surgery,
injection, catheter, suppository, or implant (e.g., implants formed
from porous, non-porous, or gelatinous materials, including
membranes, such as silastic membranes or fibers), and the like.
[0204] In a specific embodiment, one or more of the disclosed
anti-protease peptides may be associated either by coating or
impregnating an implant such as stent, for instance to treat a
dyslipidemic or vascular disorder. These peptides are prepared and
purified as described herein. In an example, the implant can be
partially or completely coated with the peptide. For instance, the
luminal surface of the implant may be coated with the peptide. Such
configuration is believed to reduce atherosclerotic plaques in
arteries often associated with atherosclerosis while minimizing the
amount of coating material and time required to prepare the
implant. The peptide may be attached to the implant by any chemical
or mechanical bond or force, including linking agents.
Alternatively, the coating may be directly linked (tethered) to the
first surface, such as through silane groups. In other examples,
the implant may be impregnated with at least one peptide by methods
known to those of skill in the art so that multiple surfaces (such
as the outer and inner surfaces) of the implant include the
peptide.
[0205] In an additional embodiment, the implant may be coated or
impregnated with materials in addition to the disclosed peptides to
further enhance their bio-utility. Examples of suitable coatings
are medicated coatings, drug-eluting coatings, hydrophilic
coatings, smoothing coatings.
[0206] The amount of the pharmaceutical compositions that will be
effective depends on the nature of the disorder or condition to be
treated, as well as the stage of the disorder or condition.
Effective amounts can be determined by standard clinical
techniques. The precise dose to be employed in the formulation will
also depend on the route of administration, and should be decided
according to the judgment of the health care practitioner and each
subject's circumstances. An example of such a dosage range is 0.1
to 200 mg/kg body weight in single or divided doses. Another
example of a dosage range is 1.0 to 100 mg/kg body weight in single
or divided doses.
[0207] The specific dose level and frequency of dosage for any
particular subject may be varied and will depend upon a variety of
factors, including the activity of the specific compound, the
metabolic stability and length of action of that compound, the age,
body weight, general health, sex, diet, mode and time of
administration, rate of excretion, drug combination, and severity
of the condition of the subject undergoing therapy.
[0208] The pharmaceutical compositions of the present disclosure
can be administered at about the same dose throughout a treatment
period, in an escalating dose regimen, or in a loading-dose regime
(e.g., in which the loading dose is about two to five times the
maintenance dose). In some embodiments, the dose is varied during
the course of a treatment based on the condition of the subject
being treated, the severity of the disease or condition, the
apparent response to the therapy, and/or other factors as judged by
one of ordinary skill in the art. The volume of administration will
vary depending on the route of administration. Those of ordinary
skill in the art will know appropriate volumes for different routes
of administration (for example, exemplary delivery methods include,
but are not limited to, those provides by Malik et al., "Recent
Advances in Protein and Peptide Drug Delivery Systems" J. Curr.
Drug Deliv. 4(2): 141-151, 2007 which is hereby incorporated by
reference in its entirety).
X. Pharmaceutical Compositions and Uses Thereof
[0209] The peptides or peptide-enhanced lipoproteins of the
disclosure (and mixtures thereof) can be used to treat any disorder
in animals, especially mammals (e.g., humans), for which inhibiting
a target protease is beneficial. Appropriate conditions will be
dependent on the protease inhibitor component used in the peptide,
and in fact the antiprotease peptide molecule is designed (and the
protease inhibitor component chosen) with the end use in mind.
Thus, for instance, conditions involving an over-abundance or
undesired activity of elastase (including A1AT deficiency) are
treated using a peptide that includes an elastase inhibitor
component. Other proteases mentioned herein have art-recognized
implications in diseases, such as inflammatory diseases, including
atherosclerosis and cancer.
[0210] The peptides, peptide analogs, and loaded lipoprotein
particles (such as HDL particles) can be used alone or in
combination therapy with other therapeutic compositions or drugs
used to treat the foregoing conditions. Such combination therapies
include, but are not limited to simultaneous or sequential
administration of the drugs involved. For example, in the treatment
of hypercholesterolemia or atherosclerosis, the peptide, peptide
analog, or lipoprotein formulations can be administered with any
one or more of known cholesterol lowering therapies, for example,
bile-acid resins, niacin, CETP inhibitors, reconstituted HDL
therapy, fibrates, PCSK9 related therapies, and statins.
[0211] In another embodiment, it may be desirable to administer the
pharmaceutical compositions locally to the area in need of
treatment. This may be achieved by, for example, and not by way of
limitation, local or regional infusion or perfusion during surgery,
topical application (e.g., wound dressing), injection, catheter,
suppository, or implant (e.g., implants formed from porous,
non-porous, or gelatinous materials, including membranes, such as
silastic membranes or fibers), and the like. Weekly or bi-weekly
administration via intravenous infusion or subcutaneous injection
is contemplated for representative embodiments.
[0212] In one embodiment, administration can be by direct injection
at the site (or former site) of a tissue that is to be treated,
such as the heart or the peripheral vasculature, lung, and so
forth. In another embodiment, the pharmaceutical compositions are
delivered in a vesicle, in particular liposomes (see, e.g., Langer,
Science 249:1527-1533, 1990; Treat et al., in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler (eds.), Liss, N.Y., pp. 353-365, 1989).
[0213] In yet another embodiment, the pharmaceutical compositions
can be delivered in a controlled release system. In one embodiment,
a pump can be used (see, e.g., Langer Science 249:1527-1533, 1990;
Sefton Crit. Rev. Biomed. Eng. 14:201-240, 1987; Buchwald et al.,
Surgery 88:507-516, 1980; Saudek et al., N. Engl. J. Med.
321:574-579, 1989). In another embodiment, polymeric materials can
be used (see, e.g., Ranger et al., Macromol. Sci. Rev. Macromol.
Chem. 23:61-64, 1983; Levy et al., Science 228:190-192, 1985;
During et al., Ann. Neurol. 25:351-356, 1989; and Howard et al., J.
Neurosurg. 71:105-112, 1989). Other controlled release systems,
such as those discussed in the review by Langer (Science
249:1527-1533, 1990), can also be used.
XI. Kits
[0214] The peptides, peptide analogs, and lipoprotein particles
disclosed herein can be supplied in the form of a kit for use in
prevention and/or treatment of diseases (e.g., a protease
inhibitor-deficiency). In such a kit, a clinically effective amount
of one or more of the peptides or peptide-loaded/enhanced
lipoprotein particles is provided in one or more containers. The
peptides or particles may be provided suspended in an aqueous
solution or as a freeze-dried or lyophilized powder, for instance.
In certain embodiments, the peptides will be provided in the form
of a pharmaceutical composition.
[0215] Kits can also include instructions, usually written
instructions, to assist the user in treating a disease (e.g., such
as a protease inhibitor deficiency) with a lipoprotein-targeting
protease inhibitor peptides. Such instructions can optionally be
provided on a computer readable medium.
[0216] The container(s) in which the protein(s) are supplied can be
any conventional container that is capable of holding the supplied
form, for instance, microfuge tubes, ampoules, or bottles. In some
applications, chimeric proteins may be provided in pre-measured
single use amounts in individual, typically disposable, tubes or
equivalent containers.
[0217] The amount of a peptide (or peptide loaded lipoprotein)
supplied in the kit can be any appropriate amount, depending for
instance on the market to which the product is directed. For
instance, if the kit is adapted for research or clinical use, the
amount of each peptide or peptide loaded lipoprotein provided would
likely be an amount sufficient for several treatments.
[0218] The following examples are provided to illustrate certain
particular features and/or embodiments. These examples should not
be construed to limit the invention to the particular features or
embodiments described.
EXAMPLES
Example 1: Rosuvastatin Alters the Proteome of High Density
Lipoproteins: Generation of Alpha-1-antitrypsin (A1AT) Enriched
Particles with Anti-Inflammatory Properties
[0219] At least some of the research described in this Example was
published Oct. 19, 2015 as Gordon et al. (Mol &Cell Proteomics
14:3247-3257, 2015), which is incorporated herein by reference in
its entirety including supplemental material.
[0220] Statins lower plasma cholesterol by as much as 50%, thus
reducing future cardiovascular events. However, the physiological
effects of statins are diverse and not all are related to LDL-C
lowering.
[0221] This Example describes a small clinical pilot study which
assessed the impact of statins on lipoprotein-associated proteins
in healthy individuals (n=10) with normal LDL-C(<130 mg/dL), who
were treated with the statin rosuvastatin (available commercially
as Crestor.RTM.) (20 mg/day) for 28 days. Proteomic analysis of
size-exclusion chromatography isolated LDL, HDL-L (large) and
HDL-S(small) fractions and spectral counting was used to compare
relative protein detection before and after statin therapy.
Significant protein changes were found in each lipoprotein pool and
included both increases and decreases in several proteins involved
in lipoprotein metabolism, complement regulation, and acute phase
response.
[0222] The most dramatic effect of the rosuvastatin treatment was
an increase in alpha-1-antitrypsin (A1AT) spectral counts
associated with HDL-L particles. Quantitative measurement by ELISA
confirmed an average 5.7-fold increase in HDL-L associated A1AT.
Molecular modeling predictions indicated that the hydrophobic
reactive center loop of A1AT, the functional domain responsible for
its protease inhibitor activity, is likely involved in its lipid
binding and its association with HDL was found to protect A1AT
against oxidative inactivation. Cell culture experiments, using
J774 macrophages, demonstrated that the association of A1AT with
HDL enhances its anti-protease activity, preventing elastase
induced production of tumor necrosis factor alpha
(TNF-.alpha.).
[0223] Thus, this Example demonstrates that statins can
significantly alter the protein composition of both LDL and HDL.
Further, described here is a novel functional relationship between
A1AT and HDL: the upregulation of A1AT on HDL enhances its
anti-inflammatory functionality, which may contribute to the
non-lipid lowering beneficial effects of statins.
Experimental Procedures
Subject Selection.
[0224] Fasting blood samples were obtained from ten healthy
volunteers participating in a study of the NIH Center for Human
Immunology, intended to evaluate effects of a statin on the immune
system. This protocol was approved by the institutional review
board of the National Heart, Lung, and Blood Institute (NHLBI) and
registered at clinicaltrials.gov (NCT01200836); all participants
provided written informed consent. Three males and seven females
were enrolled, with an average age of 44.1.+-.11 years.
Participants were selected to have normal LDL-C (<130 mg/dL) and
were not on any lipid modification therapy prior to the study.
Recruited participants were given rosuvastatin (20 mg/day) for 28
days. Blood was collected by venipuncture at the following time
points: baseline, 14 and 28 days after rosuvastatin treatment, and
14 days after stopping treatment (washout). Samples were stored at
-20.degree. C. Because samples were taken from each participant at
baseline and on-treatment, each subject acted as their own
control.
Lipoprotein Analysis.
[0225] Lipid and lipoprotein assays were performed on a Siemens
Dimension Vista analyzer, using standard enzymatic assays. HDL-C
was measured by a direct assay (Siemens) and LDL-C was determined
by the Friedewald equation. Lipoprotein particle numbers and
average particle sizes were determined from heparinized plasma on a
Vantera Clinical Analyzer (LipoScience).
Lipoprotein Isolation by Size Exclusion Chromatography.
[0226] Collected plasma from each subject at baseline (n=10) and
after 28 days of rosuvastatin treatment (n=10) was applied to two
Superdex 200 columns (GE Healthcare) arranged in series on an Akta
FPLC system. The flow rate was set to 0.5 mL/min and 0.5 mL
fractions were collected. Fractions were assayed for
phosphatidylcholine, total cholesterol, free cholesterol and
triglyceride by enzymatic assays (Wako Diagnostics) to determine
position of elution for lipoproteins. For each subject, fractions
were combined to make LDL (elution vol. 17.5-20 mL), HDL-large
(HDL-L; elution vol. 21-23 mL) and HDL-small (HDL-S; elution vol.
23-25 mL) pools, generating a total of 60 samples for MS
analysis.
Lipoprotein Proteomics--Experimental Design and Statistics.
[0227] Pooled FPLC fractions were applied to a phospholipid binding
resin and washed to isolate lipid bound protein components, as
previously described (Gordon et al., J Proteome Res 9: 5239-5249,
2010). Resin bound proteins were then subjected to overnight
trypsin digestion at 37.degree. C. Resulting peptides were
collected and then reduced with dithiothreitol (200 mM; 30 min at
37.degree. C.) and carbamidomethylated with iodoacetamide (800 mM;
30 min at 25.degree. C.). Digest solutions were dried,
reconstituted in 100 .mu.L of water+0.1% formic acid and desalted
using ZipTips (Millipore), and stored at -20.degree. C. until MS
analysis.
[0228] Desalted samples were dried and reconstituted in 20 .mu.L of
water+0.1% formic acid and 10 .mu.L was analyzed on a Thermo
Orbitrap Velos Mass Spectrometer instrument. Blank runs were
performed between each sample to prevent carry over. Peak lists
were generated using Proteome Discoverer (version 1.3.0.339) and
resulting spectra were searched against the SwissProt database
(version 012214), using Mascot (version 2.4.0) to identify protein
components of the lipoprotein fractions (fragment ion mass
tolerance of 0.80 Da and a parent ion tolerance of 20 PPM). Search
criteria included: human taxonomy, fixed modification:
carbamidomethylation (C), variable modifications: oxidation (M),
deamidation (N, Q) and up to two missed trypsin cleavage sites were
allowed. Validation of peptide and protein identifications was
performed using Scaffold software (version 4.1.1) and a 1.0% false
discovery rate (FDR) for both peptide and protein thresholds and a
minimum of 2 identified peptides were required per protein.
Calculated decoy FDR for peptide and protein identifications were
0.03% and 0.7%, respectively. Spectral counting was used as a
semi-quantitative comparison of protein abundance between baseline
and on-treatment samples using normalized spectrum count calculated
by the Scaffold software. Comparisons were only performed to
estimate the relative abundance of the same protein in the same
lipoprotein pool before and after rosuvastatin treatment; no
comparisons were made between lipoprotein fractions. Data was
analyzed by student's T-test to identify proteins with
statistically significant (p<0.05) changes in normalized
spectral counts. Because this MS analysis was intended primarily
for screening purposes, to identify candidates for hypothesis
driven functional experiments, the data analysis was not corrected
for multiple comparisons. All MS data have been deposited to the
ProteomeXchange Consortium (Vizcaino et al., Nat Biotechnol 32:
223-226, 2014) via the PRIDE partner repository with the dataset
identifier PXD002633.
Preparation of alpha-1-antitrypsin enriched HDL.
[0229] Reconstituted HDL was prepared by cholate dialysis method,
using human purified apolipoprotein A-I and
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (Avanti) as
previously described (Matz & Jonas, J Biol Chem 257: 4535-4540,
1982). Native human HDL was isolated from plasma by sequential
density gradient ultracentrifugation (Chapman et al., J Lipid Res
22: 339-358, 1981). Reconstituted or native HDL was co-incubated
with alpha-1-antitrypsin protein (Human, Sigma Aldrich) overnight
at 37.degree. C. Unbound A1AT was removed by filtration, using
Amicon Ultra 100 kDa centrifugal filter units (Millipore). Elastase
activity assays were performed with the EnzChek.RTM. elastase assay
kit (Life Technologies).
Cell Culture Experiments.
[0230] J774 mouse macrophages were used to examine the potential
functional role of alpha-1-antitrypsin enriched HDL in
inflammation. For these experiments, cells were plated in 12-well
culture plates at a density of 1.times.10.sup.5 cells/well two days
prior to the experiment. Cells were washed twice with PBS and
placed in serum-free media for 1 hour prior to addition of
treatment. Treatments (PBS, nHDL, A1AT-HDL or A1AT) were added to
serum-free culture media: HDL protein was matched for nHDL and
A1AT-nHDL at a final concentration of 200 .mu.g/mL. A1AT
concentration was matched between free A1AT and A1AT-nHDL at a
final concentration of 2 Cells were then incubated with elastase
(porcine pancreas, Sigma Aldrich, 500 nM) for 4 hours and media was
collected and centrifuged at 3,000.times.g for 5 min to pellet any
loose cells or debris and supernatant was transferred to fresh
tubes and stored at -80.degree. C. until further analysis. Elisa
assays for mouse TNF-.alpha. (BioLegend) were performed on cell
culture media.
Results
Effect of Rosuvastatin on Lipoprotein Lipid Composition and
Particle Numbers.
[0231] Serum samples from subjects at baseline and after 28 days of
rosuvastatin therapy were analyzed for plasma lipids (FIG. 1A). As
expected, treatment with rosuvastatin resulted in reductions in
total cholesterol (-28%; p<0.001), LDL-C (-50%; p<0.001) and
triglyceride (-29%; p<0.01) (FIG. 1A). There was a modest 3.6%
increase of HDL-C that was not statistically significant, although
this effect of statins has been confirmed in larger studies
(McTaggart & Jones, Cardiovasc Drugs Ther 22: 321-338, 2008).
The effect of rosuvastatin on lipoprotein particle numbers mirrored
the effects on the lipid levels. LDL particle number (LDL-p) was
reduced by 51% (p<0.001), which was predominantly due to
reduction of large LDL (FIG. 1B). HDL-p was increased by 6.2% but
was not statistically significant (FIG. 1C).
[0232] Serum was separated by gel exclusion chromatography to
isolate lipoproteins by size. Collected fractions were analyzed for
total cholesterol, free cholesterol, phosphatidylcholine, and
triglycerides (FIG. 2). Results from this analysis, indicate a
reduction in lipids associated with the LDL peak (Elution
volume=17-20 mL). This is consistent with the clinical lipid
measures presented in FIG. 1. As before, no major effect on HDL
lipids (Elution volume=21-25 mL) was observed.
Mass Spectrometry Analysis of Lipoprotein Proteome.
[0233] To test our hypothesis that rosuvastatin alters the
lipoprotein proteome, we pooled fractions containing LDL and HDL
and used mass spectrometry (MS) to analyze the lipid associated
protein content of each of these lipoproteins in each subject at
baseline and after 28 days on rosuvastatin. The HDL peak was
divided into two halves, representing large and small HDL, to gain
additional insight given the proteomic complexity of HDL. Spectral
counting was used as a semi-quantitative screen to identify
proteins whose abundance changed due to rosuvastatin treatment, an
experimental approach that has been validated in previous studies
(Gordon et al., J Proteome Res 9: 5239-5249, 2010; Gordon et al.,
Diabetes 62: 2958-2967, 2013). As additional validation of the
spectral counting approach, we also determined the correlation
between spectral counts for apolipoprotein B and LDL-p. The
stoichiometry on LDL is one molecule of apoB per particle so a
linear relationship would be expected between a quantitative
measure of apoB and LDL particle number, which was observed in FIG.
3 (R=0.884, p<0.0001).
[0234] A total of 154 proteins were identified by MS analysis of
all samples. The large number of identified proteins and the
relatively small sample size of this study resulted in few
statistically significant results after correction for multiple
comparisons; however, because the objective of this study was
strictly focused on screening to identify candidates for hypothesis
driven studies, statistical comparisons between spectral counts
from baseline and rosuvastatin treated samples were performed
without correction for multiple comparisons. In the LDL fraction,
nine proteins were found to display changes in abundance by
spectral counting as a result of the rosuvastatin treatment (FIG.
4A). The majority of these were decreases, indicating reductions in
total protein mass of LDL from the treatment. Additionally, the
magnitude of these reductions in spectral counts ranged from -20 to
-71%, which is similar to the degree of LDL particle number
lowering achieved in this study and suggests that these results are
largely a result of the LDL lowering effect of the rosuvastatin. It
is interesting, however, that complement C3 (+71%) and Ig kappa
chain V-II (+68%) demonstrated increased spectral counts in the LDL
fraction, despite the significant reduction in LDL particle number
and apoB. This relationship has not been previously described nor
is the functional importance of these changes known.
[0235] Although rosuvastatin had minimal effect on HDL lipids and
caused only a small increase in HDL particle number, several
changes were observed in the HDL proteome (FIG. 4A). Most notably,
on the large HDL, there was a marked elevation in
alpha-1-antitrypsin (A1AT) spectral counts (+2438%). In addition,
ceruloplasmin also showed an increase in spectral counts (+27%).
Reductions in spectral counts were also observed for the following
proteins: several immunoglobulin chains (variable),
N-acetylmuramoyl-L-alanine amidase (-69%), kallikrein (-26%),
clusterin (-13%) and Ig lambda like polypeptide 5 (-12%). Overall,
there were fewer changes in spectral counts in the small HDL
fraction: (alpha-1-antichymotrypsin (-89%), complement C2 (-44%),
apolipoprotein C-III (-13%) and vitronectin (+23%).
Quantitation of Alpha-1-Antitrypsin Raising Effect of
Rosuvastatin.
[0236] Of the numerous changes in the HDL proteome following
rosuvastatin treatment, we focused on A1AT because it showed the
largest change and the function of A1AT is relatively well
understood. In fact, the magnitude of the increase of HDL bound
A1AT in this study was much greater than changes found in previous
studies that examined the effect of various diseases or therapy on
lipoprotein proteome (Vaisar et al., J Clin Invest 117: 746-756,
2007; Gordon et al., Diabetes 62: 2958-2967, 2013; Green et al.,
Circulation 118: 1259-1267, 200). Additionally, A1AT has been shown
to be involved in cardiovascular disease by modulating various
inflammatory processes (Gilutz et al., Br Heart J 49: 26-29, 1983;
Duckers et al., Respir Res 11: 173, 2010).
[0237] To more quantitatively evaluate the change in A1AT levels
observed in the MS data, we performed ELISA assays to measure A1AT
in the large HDL fractions from all subjects. There was a distinct
segregation of the subjects into two groups: high responders that
showed a marked elevation of A1AT (range: 5-18 fold) in their large
HDL fraction in response to rosuvastatin therapy and low-responders
that only showed a modest increase in A1AT levels (range: 6-36%)
after therapy. This segregation was consistent with our MS data
(FIGS. 5A and 5B) and did not appear to be associated with baseline
plasma lipid measures or C-reactive protein (CRP).
[0238] In addition to measuring the A1AT content of large HDL, we
also measured total A1AT in plasma. There was a clear trend of
increasing plasma A1AT while on treatment and a return to baseline
after a two week treatment washout, this effect was relatively
small, approximately 10% (FIG. 5c). The change in total A1AT did
not appear to correlate with the change observed in A1AT on large
HDL after rosuvastatin therapy. Based on the content of A1AT on
HDL-L and in plasma, we estimate that on average approximately
4.54.+-.1.73% of total A1AT was bound to HDL after rosuvastatin
therapy, whereas only 0.84.+-.0.037% was bound at baseline before
treatment. We calculated an A1AT:HDL-particle ratio (mol:mol) of
about 1:100 at baseline and 1:25 after rosuvastatin treatment.
A1AT Binds to Lipids with its Reactive Center Loop.
[0239] To examine the potential structural basis for the
interaction of A1AT with lipoproteins, we used modeling software
that calculated the optimum thermodynamic orientation for a protein
to interact with a phospholipid bilayer, using their 3D structures.
The crystal structure for A1AT was downloaded from Protein Data
Bank (available on the World Wide Web at rcsb.org; structure ID:
3NE4) and used as input for the lipid binding prediction in the
position of proteins in membranes server (available online at
opm.phar.umich.edu/) (Lomize et al., Nucleic Acids Res 40:
D370-376, 2012). From this analysis, an exposed random coil region
of the A1AT protein was predicted to insert into the outer leaflet
of a phospholipid bilayer (FIG. 6A). This region of A1AT
corresponds to the reactive center loop (RCL) of A1AT, which
contains the active site responsible for the ability of this
protein to inhibit proteolytic activity, and is relatively
hydrophobic. In this binding model, two methionine residues
critical to the function of A1AT (Met351 & Met358) are
predicted to be buried in the lipid surface. These methionine
residues are highly susceptible to oxidation to methionine
sulfoxide. Previous studies have shown that oxidative modification
of either of these residues will result in the loss of
anti-elastase activity (Taggart et al., J Biol Chem 275:
27258-27265, 2000). If the RCL region of the protein is removed
from the protein structure (A1AT.DELTA.346, FIG. 6B), the protein
is predicted to have markedly reduced affinity for the lipid
surface, as indicated by decreased calculated insertion depth and
decreased .DELTA.G.sub.transfer energy.
[0240] Based on this lipid binding model for A1AT, we hypothesized
that the burying of the RCL domain into the lipids surface of HDL
would affect its capacity to inhibit elastolytic activity. To test
this, we made reconstituted HDL containing A1AT by cholate
dialysis. As can be seen by the gel filtration profile, when A1AT
was incubated with discoidal HDL, containing apoA-I and
phospholipids, a significant fraction of A1AT associates with HDL
(FIG. 7A). Similarly, and consistent with previous studies, when
native HDL isolated from plasma was incubated with A1AT, it became
enriched in A1AT (Moreno et al., Am J Respir Cell Mol Biol 51:
536-549, 2014).
[0241] Next, we compared the ability of A1AT bound to HDL versus
free A1AT to inhibit elastase activity. The HDL particle bound A1AT
showed a 5-fold reduction in elastase inhibitor capacity (FIG. 7B),
suggesting that the active site in the RCL is sterically blocked
from interacting with elastase when bound to HDL. Overall, this
data supports our model of lipid binding and suggests several
possible functional implications of the interaction between A1AT
and HDL, which we tested below.
HDL Binding Protects A1AT Anti-Elastase Activity from Inactivation
by H.sub.2O.sub.2.
[0242] In atherosclerotic plaque, activated neutrophils and
macrophages create an oxidizing environment by generating reactive
oxygen species, such as hydrogen peroxide (H.sub.2O.sub.2) (Nathan
& Root, J Exp Med 146: 1648-1662, 1977; Rajagopalan et al., J
Clin Invest 98: 2572-2579, 1996). Based on the binding model of
A1AT to HDL, we predicted that submersion of the critical Met351
& Met358 residues, which are susceptible to H.sub.2O.sub.2
oxidation, in the lipid surface may confer protection of these
residues in the RCL against oxidation and therefore promote the
preservation of anti-elastase activity in an oxidizing environment.
To test this hypothesis, we generated A1AT enriched native HDL
(nHDL) by co-incubating A1AT with isolated human HDL and then
removed unbound A1AT. Either free A1AT or A1AT-nHDL was exposed to
H.sub.2O.sub.2 at various concentrations and remaining
anti-elastase activity was measured. Nonlinear regression analysis
of the dose response curves of anti-elastase activity versus
[H.sub.2O.sub.2] indicated a shift in IC.sub.50 (28.09 vs 46.42 mM
H.sub.2O.sub.2 for A1AT and A1AT-HDL, respectively; p<0.0001),
indicating that HDL bound A1AT was more resistant to inactivation
by H.sub.2O.sub.2 (FIG. 8). This finding suggested that although
binding of A1AT to reconstituted HDL resulted in a reduction of
A1AT activity, the interaction of A1AT with HDL may be beneficial
in stabilizing its activity in a pro-oxidant environment, such as
an atherosclerotic plaque.
Effects of A1AT Enriched HDL on Elastase Induced Macrophage
Activation.
[0243] An early event in the initiation of atherosclerosis is the
infiltration of circulating neutrophils and monocytes into the
sub-endothelial space. Upon activation, neutrophils and macrophages
in this environment produce oxygen radicals and also secrete
neutrophil elastase, a proteolytic enzyme, which can cause
degradation of the extracellular matrix and activation of
proinflammatory signaling pathways in nearby cells via protease
activated receptors. We used the J774 macrophage cell line to
examine the proinflammatory response induced by elastase exposure
and to determine the effect A1AT enriched HDL would have on this
response.
[0244] We first demonstrate that, in the J774 cell system, exposure
to elastase induces a strong proinflammatory cytokine response,
with increased TNF-.alpha. in the cell media in a dose-dependent
manner and this effect is absent when treated with heat-inactivated
elastase (FIG. 9A). Next, J774 cells were pre-incubated with PBS,
native HDL (nHDL), A1AT enriched nHDL, or lipid-free A1AT for 1
hour prior to the addition of elastase to the culture media to
allow for inactivation of A1AT. Upon elastase addition, cells
pretreated with PBS displayed a robust production of TNF-.alpha..
Native HDL had a strong anti-inflammatory effect, significantly
reducing TNF-.alpha. production by -77.2% compared to PBS
pretreatment (FIG. 9B). This already impressive effect was further
amplified and TNF-.alpha. production was almost completely
suppressed (-97%) when cells were pretreated with HDL further
enriched in A1AT. In contrast, pretreatment with lipid-free A1AT
had no significant protection against elastase induced TNF-.alpha.
production. The almost complete lack of effect with lipid-free A1AT
was unexpected but suggests that in the lipid-free form, A1AT may
be quickly degraded or inactivated when cultured with
macrophages.
[0245] To determine if degradation was responsible for the loss of
A1AT activity, we performed western blot for A1AT on the cell
culture media and found no evidence of proteolytic degradation of
A1AT. This suggested to us that the loss of anti-protease activity
in the lipid-free A1 AT treatment may be due to another
inactivating protein modification. We then performed elastase
inhibition assays on each of the treatments in FIG. 9B, but in a
cell-free system. The results of this experiment demonstrated that
the PBS and nHDL treatments did not inhibit elastase activity,
however, both A1AT HDL and lipid-free A1AT showed significant
anti-elastase activity, indicating that the cell culture
environment inactivated the lipid-free A1AT but did not affect the
HDL bound A1AT (FIG. 9C). In further support of this model, when
each of the treatments was pre-incubated with elastase prior to
addition to the cells, free A1AT was then readily able to inhibit
elastase induced TNF-.alpha. production (FIG. 9D).
[0246] In another experiment, J774 cells were pretreated in the
same manner as for FIG. 9B, except that the cell media was removed
and the cells were washed prior to addition of fresh serum free
media containing elastase. Under these conditions, the nHDL
pretreatment still provided significant protection against elastase
induced TNF-.alpha. production (FIG. 9E), indicating that the
effect of native HDL is occurring via an intracellular or signaling
pathway. However, the additional protection conferred by A1AT
enrichment was lost, indicating that the mechanism of action for
A1AT HDL is by direct inhibition of elastase in the culture media.
Treatment with lipid-free A1AT still had no effect.
[0247] Overall, this data supports a mechanism whereby lipid-free
A1AT is rapidly inactivated in macrophage culture media and is
unable to inhibit elastase induced cytokine production by
macrophage cells. However, when associated with HDL, A1AT retains
its anti-elastase activity and efficiently inhibits elastase
induced TNF-.alpha. production. Based on our lipid binding model,
we predict that the preservative effect of HDL binding on A1AT
activity may be a result of the interaction of the RCL with the
lipid surface conferring protection in an oxidizing
environment.
Discussion
[0248] In this Example, we report the impact of rosuvastatin
treatment on the lipoprotein proteome in humans and found that
several protein components of both LDL and HDL particles were
affected by treatment. The majority of proteome effects on LDL were
reductions in protein content, which likely reflect the overall
reduction in LDL particle number associated with statin therapy.
However, despite this reduction in particle number, there were two
proteins that displayed increased abundance on LDL with statin
treatment, namely complement C3 and Ig kappa chain V-II. We found
no existing reports of direct functional relationships between
these proteins and LDL, but their elevation on the background of
dramatic LDL lowering may indicate the presence of a subpopulation
of LDL that is either resistant to lowering by rosuvastatin or
perhaps is generated as a consequence of the treatment. Whether or
not these particles have any influence on lipoprotein metabolism or
cardiovascular disease is an area of interest for future
studies.
[0249] Although rosuvastatin treatment had little or no impact on
HDL particle number, total HDL-C, or HDL lipid profile by FPLC,
there were several major proteome changes observed. Among these was
a dramatic 24-fold increase in A1AT spectral counts associated with
large HDL, a protein change much greater than typically observed in
other similar lipoprotein proteome studies. Because of this and the
known function of A1AT, we chose to focus our attention on this
protein change and to investigate its functional consequences. A1AT
is an acute phase reactive plasma protein with a typical plasma
concentration below 1.5 mg/mL. It belongs to the serine protease
inhibitor (SERPIN) family of proteins of which there are 36 members
in humans, 29 of which have anti-protease activity. A1AT is one of
the most abundant SERPINs and is the primary physiological
inhibitor of neutrophil elastase (NE) (Gettins, Chem Rev 102:
4751-4804, 2002). NE is produced by activated neutrophils and
macrophages in atherosclerotic lesions where it degrades components
of the extracellular matrix (i.e. elastin, collagen and
fibronectin) (Weiss, N Engl J Med 320: 365-376, 1989; Dollery et
al., Circulation 107: 2829-2836, 2003). Additionally, cholesterol
loaded monocyte-derived macrophages, such as those found in
atherosclerotic plaque, express elevated elastolytic activity
(Rouis et al., Arteriosclerosis 10: 246-255, 1990). Besides
degrading extracellular matrix, elastase can also stimulate
production of pro-inflammatory cytokines and trigger pro-apoptotic
signaling via protease activated receptors expressed on most cells
(Shpacovitch et al., J Leukoc Biol 83: 1309-1322, 2008;
Ramachandran et al., J Biol Chem 286: 24638-24648, 2011). These
combined activities of elastase within the vessel wall likely
contribute to atherosclerotic progression by promoting smooth
muscle cell migration, endothelial cell apoptosis, and plaque
instability (Garcia-Touchard et al., Arterioscler Thromb Vasc Biol
25: 1119-1127, 2005).
[0250] It has been demonstrated that HDL is actively transported
across the vascular endothelium at the site of atherosclerotic
plaque (Rohrer et al., Circ Res 104: 1142-1150, 2009), thus
delivering HDL to this inflammatory, proteolytic environment. The
fate of the protein cargo (such as non-apoA-I proteins) of HDL
during this process has not yet been examined. However, it seems
possible that the process of endothelial transcytosis of HDL may
act as a mechanism for delivery of HDL's largely anti-inflammatory
and anti-proteolytic cargo to a site of inflammatory distress. A1AT
is fairly abundant in plasma, but it may need to reach the
sub-endothelial space, where elastolytic proteases are produced by
activated neutrophils and macrophages, to effectively inhibit
protease mediated damage to the vessel wall. Although some small
plasma proteins are capable of relatively rapid movement across the
vascular endothelium, A1AT is similar to albumin in size and
negative charge (pI.apprxeq.4), and thus it is likely not capable
of passive diffusion across the endothelium and may require active
transport or a chaperone such as HDL.
[0251] Our findings expand significantly upon existing studies by
first showing that A1AT binding to HDL is induced by rosuvastatin
and by identifying a previously unidentified functional benefit of
this association. Our data support a protective role for HDL,
whereby the anti-elastase activity of A1AT is preserved by
interaction with HDL. Although our experiments with reconstituted
HDL demonstrate that A1AT activity is reduced upon binding to HDL,
a significant amount of anti-protease activity remains.
Furthermore, it has recently been shown, in atherosclerotic
plaques, that HDL particles eventually disassemble when they enter
tissue and therefore may release free A1AT (Huang et al., Nat Med
20: 193-203, 2014). Based on our HDL binding model and our
experiments with hydrogen peroxide, one possible mechanism for the
preservation of A1AT activity when bound to HDL may be through
shielding of oxidation susceptible methionine residues in the RCL,
which when oxidized result in loss of A1AT activity. Other possible
mechanisms may involve the protection of A1AT from degradation by
other macrophage secreted proteases or HDL-directed cellular uptake
and intracellular anti-proteolytic activity. Detailed mechanistic
and structural investigations of these hypotheses are underway.
[0252] The association of A1AT with both LDL and HDL particles has
been previously demonstrated by biochemical and proteomics based
techniques. Binding to LDL occurs when A1AT has been oxidized and
is inactivated (Mashiba et al., Arterioscler Thromb Vasc Biol 21:
1801-1808, 2001), but a change in the oxidation status of A1AT has
not been previously implicated in altering its binding to HDL.
There have been no reports on the mechanism of A1AT binding to HDL.
It is currently unclear whether A1AT becomes associated with HDL
during particle generation or is in a state of equilibrium,
constantly exchanging to and from preformed HDL particles. Our data
suggest only a minimal increase in total plasma A1AT while on
rosuvastatin, an amount that is likely insufficient to account for
a simple equilibrium driven shift onto HDL of the magnitude seen in
this study. This suggests the possibility of increased de novo
generation of A1AT containing HDL or that an additional plasma
factor or a change in some physical property of HDL in response to
statin treatment that may be responsible for the increased binding
of A1AT. There have been only a limited number of functional
investigations of A1AT on HDL. Studies by Meilhac et al first
suggested a possible functional role for HDL bound A1AT in
preventing apoptosis in vascular smooth muscle cells (Ortiz-Munoz
et al., FASEB J 23: 3129-3139, 2009). A second study from the same
group demonstrated a role for HDL in the selective transport of
functional A1AT from the circulation to the lung, where it can
prevent elastase induced morphological and functional damage in a
rat model (Moreno et al., Am J Respir Cell Mol Biol 51: 536-549,
2014). These findings suggest a possible important role of A1AT
bound HDL for the treatment of chronic obstructive pulmonary
disease (COPD) or emphysema, where excessive elastolytic activity
causes destruction of lung tissue and is often not adequately
treated by replacement therapy with just A1AT (Abusriwil &
Stockley, Curr Opin Pulm Med 12: 125-131, 2006).
[0253] Although the focus of this study is on A1AT, nine other
members of the SERPIN family are consistently identified in
proteomics studies of HDL (Davidson, The HDL Proteome Watch.
available on-line at
homepages.uc.edu/.about.davidswm/HDLproteome.html, 2015). The
complete structural information is not available for all of these,
but all of the well characterized SERPINs have structural
organizations similar to that of A1AT with an exposed hydrophobic
meta-stable RCL domain. It may be that many of these other SERPINs
also bind HDL by a similar mechanism involving the RCL and similar
functional relationships exist for this entire class of
proteins.
[0254] Additional questions raised by the present report include
whether the observed effects of rosuvastatin on the lipoprotein
proteome are similar among all statins and whether this accounts
for some of the known anti-inflammatory effect of statins
(Antonopoulos et al., Curr Pharm Des 18: 1519-1530, 2012). One
study of the HDL proteome, in patients receiving combination
therapy of atorvastatin and niacin, found reduced apoE content in
HDL.sub.3, however no effect on A1AT was detected (Green et al.,
Circulation 118: 1259-1267, 2008). The absence of effect on A1AT in
this study may be explained by several factors including the use of
a different statin, addition of niacin treatment, or differences in
HDL isolation (ultracentrifugation vs. gel filtration). Some of the
anti-inflammatory effects of statins have been attributed to the
ability to block the Rho signaling pathway (Martin et al., J Clin
Invest 107: 1423-1432, 2001) but based on the results of this study
their ability to also modulate A1AT levels on HDL may also be
relevant. It is interesting to note that there have been several
recent reports of statins (i.e. Simvastatin and atorvastatin)
having protective effects on lung injury induced by elastase
(Takahashi et al., Am J Physiol Lung Cell Mol Physiol 294:
L882-890, 2008; Boiati et al., Drug Res (Stuttg). 65(10):540-544,
2014) or by cigarette smoke (Lee et al., Chest 128: 574S, 2005;
Wright et al., Am J Respir Crit Care Med 183: 50-58, 2011), which
could potentially also be mediated by A1AT enriched HDL.
[0255] Studies of the lipoprotein proteome have gained increasing
interest due to their clear potential for identifying functionally
relevant lipoprotein subspecies. However, the list of lipoprotein
associated proteins is growing faster than our understanding of the
functional relevance of these complexes. New insight into the
diagnostic or therapeutic implications of HDL sub-fractions with
specific protein content will likely require direct experimental
investigation as was done in this study. Although much still needs
to be done in regard to the role of A1AT interaction with HDL, to
our knowledge this is the first report of a newly identified
protein on HDL by LC-MS that has been shown to have a possible
functional role in the pathogenesis of atherosclerosis.
Example 2: Alpha-1-antitrypsin Protects High Density Lipoprotein
from Functional Inactivation by Elastase
[0256] High density lipoproteins (HDL) are complexes of lipid and
protein with several known atheroprotective functions. These
functions are driven by specific lipids and proteins contained on
the HDL particle and include reverse cholesterol transport,
suppression of inflammation, and modulation of endothelial
function. These activities are most important within
atherosclerotic plaque, a harsh environment where HDL interact with
macrophage foam cells, activated neutrophils, and dysfunctional
endothelial cells. Neutrophils and macrophages secrete proteases
such as elastase, which damage structural components and soluble
proteins and propagate inflammatory signaling. It has been
suggested that, in plaque, HDL become damaged and
dysfunctional.
[0257] We recently characterized a subspecies of HDL which carries
the protein alpha-1-antitrypsin (A1AT), an abundant plasma serine
protease inhibitor (Gordon et al., Mol &Cell Proteomics
14:3247-3257, 2015). In the current Example, we test the hypothesis
that A1AT enriched HDL are protected from proteolytic damage and
functional inactivation that is caused by elastase, the main
protease inhibited by A1AT. Human HDL was isolated by
ultracentrifugation, and was enriched with A1AT by co-incubation
then unbound A1AT was removed. Treatment of native HDL with
elastase resulted in significant proteolytic degradation of both
apoA-I and apoA-II, visualized by Coomassie stained SDS-PAGE.
Analysis of lipoprotein size by one dimensional native gel
electrophoresis revealed that pre-beta HDL were completely
destroyed by elastase. Compared to native HDL, A1AT enriched HDL
samples were protected from protein and pre-beta particle
degradation by elastase.
[0258] We next tested the effect of elastase treatment on HDL
function. In native HDL, elastase had damaging effects on ABCA1
mediated cholesterol efflux (-32%; p<0.0001) and the ability to
esterify free cholesterol (-14%; p<0.02). A1AT enriched HDL
displayed no loss of functionality upon treatment with elastase.
Both of these activities are required for HDL to perform what is
thought to be its most important function, reverse cholesterol
transport.
[0259] In conclusion, the data presented here indicate that HDL
particles which contain A1AT may represent a functionally important
species of HDL which have an advantage in the protease-rich plaque
environment.
Methods:
[0260] Isolation of Human HDL and Enrichment with
Alpha-1-antitrypsin.
[0261] HDL was isolated from human plasma by density gradient
ultracentrifugation (1.063-1.21 g/mL). Isolated HDL was enriched
with full length A1AT protein (Sigma, purified from human plasma)
by co-incubation overnight at 37.degree. C. HDL was re-purified by
size-exclusion chromatography to remove unbound A1AT.
[0262] Elastase Treatment of HDL.
[0263] Native HDL or A1AT enriched HDL were treated with various
doses of elastase (0, 0.25, or 0.5 units) for 60 min. at 37.degree.
C. To examine the effect of elastase treatment on individual HDL
proteins, samples were analyzed by SDS-PAGE and Coomassie blue
staining. Additionally, samples treated at the highest dose of
elastase (0.5 units) were analyzed by one dimensional native gel
electrophoresis to examine the effect of elastase treatment on
intact lipoprotein particles. The gel was transferred to PVDF
membrane and western blotting was used to detect apoA-I protein.
The approach allows for detection of .alpha. and pre-.beta.
lipoprotein particles.
Functional Analysis of Elastase Treated HDL.
[0264] Cholesterol Efflux.
[0265] BHK cell lines stably transfected with either MOCK
(control), ABCA1, or ABCG1 cholesterol transporters were used to
measure cholesterol efflux to elastase treated HDL. Briefly, cells
were loaded with radioactive .sup.3H labeled free-cholesterol by
co-incubation and unincorporated cholesterol was removed by
washing. Samples were added to the cell culture media and incubated
for 6 hours. After incubation, the transfer of radiolabeled
cholesterol from the cells to media was measured by scintillation
counting and the amount of cholesterol efflux was calculated.
[0266] Activation of Lecithin Cholesterol Acyltransferase
(LCAT).
[0267] The assay for LCAT activity involves incorporation of
.sup.3H-labeled free cholesterol into isolated human HDL followed
by incubation at 37.degree. C. to activate endogenous LCAT. After
incubation, lipids were extracted and thin layer chromatography was
used to separate free cholesterol from esterified cholesterol (the
product of LCAT activity). The spots for free cholesterol and
esterified cholesterol were collected and radioactivity associated
with each measured to determine their ratio, which indicates the
level of LCAT activity.
[0268] Paraoxonase (PON1) Activity.
[0269] HDL associated PON1 activity (arylesterase) was measured
using a commercially available kit (Zeptometrix) that measures the
cleavage of phenyl acetate to produce phenol. The activity was
measured by monitoring light absorbance at 270 nm.
Results:
[0270] Elastase Treatment of HDL.
[0271] FIG. 10 shows that enrichment of HDL with A1AT protects HDL
proteins (apoA-I and apoA-II) from degradation by elastase (FIG.
10A). Additionally, elastase treatment results in degradation of
pre-beta HDL and this is also protected by A1AT (FIG. 10B).
Pre-beta HDL is an important subpopulation of HDL which functions
to perform cholesterol efflux from cells via the ABCA1 transporter.
This function is believed to be one of the most important
protective functions of HDL.
[0272] Functional Analysis of Elastase Treated HDL.
[0273] We tested the effect of elastase treatment on three
different HDL functions: cholesterol efflux (FIG. 11A), PON1
Activation (FIG. 11B) and LCAT activity (FIG. 11C). In each case,
elastase treatment of HDL resulted in a reduction of activity
(first and second bars in each panel). However, A1 AT enrichment
protected HDL from elastase treatment (third and fourth bars in
each panel). A1AT enriched HDL exist naturally in the circulation
in humans but represent only about 1% of the total HDL
population.
[0274] Additionally, Example 1 indicates that statin treatment may
increase the amount of A1AT containing HDL. Overall, these data
support the concept that A1AT enriched HDL are protected from
functional inactivation which, evidence suggests, can happen within
atherosclerotic plaques, where HDL function is needed most.
Example 3: HDL Targeting Protease Inhibitor
[0275] This example describes production and characterization of a
representative lipoprotein targeting protease inhibitor fusion
molecule, referred to as E-AAPV peptide (a.k.a. vitE-PEG-AAPV; SEQ
ID NO: 5) (vitamin E linked to the A1AT linear peptide AAPV (SEQ ID
NO: 1) via a PEG.sub.2000 linker). This peptide molecule (shown
pictorially in FIG. 13A) is a mimetic of A1AT and is designed to
bind to HDL (shown pictorially in FIG. 13B) and to inhibit elastase
activity similarly to full length A1AT.
[0276] Construction of VitE-PEG.sub.2000-AAPV Peptide.
[0277] AAPV-CMK (2.5 mg; SEQ ID NO: 3) was dissolved in 0.5 ml of
0.1 M Na-Bo buffer, pH 8.9. While stirring at room temperature,
solid VitE-PEG-NHS (5-fold molar excess over AAPV-CMK) was added
stepwise into the vial. The pH was monitored and adjusted to
maintain pH 8.9 as needed, by addition of 0.5 M NaOH. See FIG. 14
for an overview of the reaction.
[0278] Assessment of Peptide Production Reaction.
[0279] Reaction progress was monitored by MALDI mass spectrometry
of ZipTip desalted samples at various points during the reaction
(FIG. 15). The PEG linker produces a mass envelope with an average
mass of 2000 daltons. The shift in the mass envelope to an average
mass of about 2300 daltons (FIG. 15, third panel) is consistent
with conjugation of the AAPV-CMK peptide. Additionally, reaction
completion was confirmed using ninhydrin reaction to indicate that
all available amines (peptide N-terminus) had been conjugated.
[0280] Elastase Inhibitor Assay.
[0281] Elastase inhibitor activity was evaluated using the
commercially available EnzChek Elastase Assay Kit (Molecular
Probes, Cat. # E-12056) and manufacturer suggested protocols. FIG.
16 shows elastase inhibitor activity of the reactants AAPV (SEQ ID
NO: 1) and VitE-PEG and the product fusion peptide, E-AAPV
(VitE-PEG.sub.2000-AAPV). This data shows that VitE-PEG molecule
alone does not convey elastase inhibition and that Vitamin E
conjugation to the AAPV peptide does not affect elastase inhibition
activity.
[0282] E-AAPV Peptide Binds HDL and Confers Elastase Inhibitor
Activity.
[0283] The reaction product (E-AAPV; SEQ ID NO: 5) was coincubated
with purified human HDL for 30 minutes at 37.degree. C. followed by
repurification of HDL by size-exclusion chromatography to remove
unbound E-AAPV peptide. The repurified HDL was then tested for
elastase inhibitor activity using the assay described above.
[0284] E-AAPV loaded HDL was compared to the same HDL that was
coincubated with buffer only (No peptide). The HDL and HDL+E-AAPV
were normalized based on phospholipid content, determined by
colorimetric assay, prior to measurement of elastase activity. FIG.
17 shows that the E-AAPV peptide binds HDL and confers
dose-dependent elastase inhibitor activity.
Discussion
[0285] Described above is the design and production of a
lipoprotein targeting protease inhibitor peptide with potential
therapeutic application, for instance, in patients with
alpha-1-antitrypsin deficiency. This design is the result of the
conjugation of two readily available components using amine
reactive chemistry (N-hydroxysuccinimide) (FIG. 14) to generate the
final product. We have demonstrated that the VitE-PEG-AAPV peptide
retains elastase inhibitor activity and that this activity will
associate with Human HDL after a brief co-incubation.
Example 4: Method of Treating A1AT Deficiency in a Subject
[0286] According to the teachings herein, one or more of the
disclosed lipoprotein targeting protease inhibitor peptide
comprising an elastase-inhibiting portion can be used to overcome,
treat or inhibit A1AT deficiency and related symptoms in a subject.
A method of overcome, treat or inhibit A1AT deficiency and related
symptoms in a subject includes administering to the subject a
therapeutically effective amount of a pharmaceutical composition
including one or more of the peptides disclosed herein. In one
example, a therapeutically effective amount of the pharmaceutical
composition is provided by injecting intravenously 30 mg/kg of one
or more of the disclosed peptides once a week. In certain
representative examples, a pharmaceutical composition includes one
of the following peptides: VitE-AAPV (SEQ ID NO: 4); VitE-PEG-AAPV
(SEQ ID NO: 5); VitE-PEG-AAPV-CMK (SEQ ID NO: 6); VitE-AAPV-CMK
(SEQ ID NO: 7); VitE-PEG-KRCCPDTCGIKCL (SEQ ID NO: 8);
VitE-PEG-KRMMPDTMGIKML (SEQ ID NO: 9); VitE-PEG-EEIIMD (SEQ ID NO:
10); VitE-PEG-hirudin; VitE-PEG-lepirudin; VitE-PEG-desirudin; SEQ
ID NO: 13; or SEQ ID NO: 14.
[0287] In another particular example, a method of treating A1AT
deficiency or a symptom associated therewith is disclosed in which
a pharmaceutical composition includes a peptide selected from among
VitE-AAPV (SEQ ID NO: 4); VitE-PEG-AAPV (SEQ ID NO: 5);
VitE-PEG-AAPV-CMK (SEQ ID NO: 6); VitE-AAPV-CMK (SEQ ID NO: 7);
VitE-PEG-KRCCPDTCGIKCL (SEQ ID NO: 8); VitE-PEG-KRMMPDTMGIKML (SEQ
ID NO: 9); VitE-PEG-EEIIMD (SEQ ID NO: 10); VitE-PEG-hirudin;
VitE-PEG-lepirudin; VitE-PEG-desirudin; SEQ ID NO: 13; and SEQ ID
NO: 14, and such composition is administered intraperitoneally at
150 mg/kg.
Example 5: Associating a Lipoprotein Targeting Protease Inhibitor
Peptide with an Implant
[0288] According to the teachings herein, one or more peptides
comprising a lipoprotein targeting domain and a protease inhibitor
domain, optionally further including therebetween a linker, can be
placed in a suitable container, such as a tissue microcapsule
implant, and placed within a subject to allow continuous, slow
release of one or more of the disclosed peptides. Such peptides can
either be provided in the free state or after complexation with
lipid (e.g., in the form of a loaded or enriched nHDL or rHDL).
Example 6: Production of Additional Lipoprotein Targeting Protease
Inhibitor Peptides
[0289] This example describes the production of additional
inhibitor peptides useful in the methods described herein.
[0290] Peptides were prepared (without C-terminal Valine-CMK) by
solid phase synthesis using standard amino acids. After addition of
the PEG, FITC (in the fluorescent labeled peptide), and VitE
entities, valine chloromethylketone (CMK) was added to C-terminus
by condensation reaction (Thompson, Biochemistry. 1973, PMID:
4734223). The following three peptides were produced:
##STR00004##
[0291] In the above formulae, (PEG).sub.2=Fmoc-NH-(PEG).sub.2-COOH
(20 atoms) (Cat. #851031, from Novabiochem.RTM.; now EMD Millipore,
Billerica, Mass.); FITC=Fmoc-K(FAM)-OH (from AnaSpec, Fremont,
Calif.), which was selectively conjugated to the e group of Lys
residue in fluorescent peptide; and VitE=Vitamin E.
[0292] This disclosure provides lipoprotein targeting protease
inhibitor peptides, compositions comprising such peptides
(including protease inhibitor enriched HDL), and methods of their
use. The disclosure further provides A1AT-enriched HDL particles,
and methods of making and using such particles. It will be apparent
that the precise details of the compositions, preparations, and
methods described may be varied or modified without departing from
the spirit of the described invention. We claim all such
modifications and variations that fall within the scope and spirit
of the claims below.
Sequence CWU 1
1
1514PRTartificial sequencesynthetic peptide 1Ala Ala Pro
Val124PRTArtificial sequenceSynthetic
peptideMOD_RES(4)..(4)modified with CH2Cl 2Ala Ala Pro
Val134PRTArtificial sequenceSynthetic
peptidemod_res(4)..(4)modified with CMK 3Ala Ala Pro
Val144PRTartificial sequencesynthetic
peptidemod_res(1)..(1)Modified with vitamin E (alpha tocopherol)
4Ala Ala Pro Val154PRTArtificial sequenceSynthetic
peptidemod_res(1)..(1)Modified with poly(ethylene) glycol modified
with vitamin E (alpha tocopherol) 5Ala Ala Pro Val164PRTArtificial
Sequencesynthetic peptidemod_res(1)..(1)Modified with
poly(ethylene) glycol modified with vitamin E (alpha
tocopherol)Mod_Res(4)..(4)Modified with CMK (Chloromethyl ketone)
6Ala Ala Pro Val174PRTArtificial SequenceSynthetic
peptidemod_res(1)..(1)Modified with vitamin E (alpha
tocopherol)mod_res(4)..(4)Modified with CMK (chloromethyl ketone)
7Ala Ala Pro Val1813PRTArtificial sequenceSynthetic
peptideMod_res(1)..(1)Modified with poly(ethylene) glycol modified
with vitamin E (alpha tocopherol) 8Lys Arg Cys Cys Pro Asp Thr Cys
Gly Ile Lys Cys Leu1 5 10913PRTArtificial SequenceSynthetic
peptidemod_res(1)..(1)Modified with poly(ethylene) glycol modified
with vitamin E (alpha tocopherol) 9Lys Arg Met Met Pro Asp Thr Met
Gly Ile Lys Met Leu1 5 10106PRTArtificial SequenceSynthetic
peptidemod_res(1)..(1)Modified with poly(ethylene) glycol modified
with vitamin E (alpha tocopherol) 10Glu Glu Ile Ile Met Asp1
51165PRTHirudo medicinalis 11Met Thr Tyr Thr Asp Cys Thr Glu Ser
Gly Gln Asn Leu Cys Leu Cys1 5 10 15Glu Gly Ser Asn Val Cys Gly Gln
Gly Asn Lys Cys Ile Leu Gly Ser 20 25 30Asp Gly Glu Lys Asn Gln Cys
Val Thr Gly Glu Gly Thr Pro Lys Pro 35 40 45Gln Ser His Asn Asp Gly
Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu 50 55 60Gln651258PRTBos
taurus 12Arg Pro Asp Phe Cys Leu Glu Pro Pro Tyr Thr Gly Pro Cys
Lys Ala1 5 10 15Arg Ile Ile Arg Tyr Phe Tyr Asn Ala Lys Ala Gly Leu
Cys Gln Thr 20 25 30Phe Val Tyr Gly Gly Cys Arg Ala Lys Arg Asn Asn
Phe Lys Ser Ala 35 40 45Glu Asp Cys Met Arg Thr Cys Gly Gly Ala 50
55138PRTArtificial SequenceSynthetic peptidemod_res(1)..(1)Modified
with poly(ethylene) glycol modified with vitamin E (alpha
tocopherol)mod_res(8)..(8)Modified with CMK (chloromethyl ketone)
13Lys Gly Ser Gly Ala Ala Pro Val1 5148PRTartificial
sequenceSynthetic peptidemod_res(1)..(1)Modified with
poly(ethylene) glycol modified with vitamin E (alpha
tocopherol)mod_res(1)..(1)Further modified with attached FITC
(Fmoc-K(FAM)-OH) groupMod_res(8)..(8)Modified with CMK
(chloromethyl ketone) 14Lys Gly Ser Gly Ala Ala Pro Val1
5154PRTArtificial SequenceSynthetic peptideMISC_FEATURE(1)..(1)X is
Arginine, Phenylglycine, or NorleucineMISC_FEATURE(2)..(2)X is
Alanine, Phenylglycine, Citrulline, or
NorleucineMISC_FEATURE(3)..(3)X is Trp, Val, or
TyrMISC_FEATURE(4)..(4)X is Phenylglycine or Glycine 15Xaa Xaa Xaa
Xaa1
* * * * *