U.S. patent application number 14/058808 was filed with the patent office on 2014-02-13 for serpin drugs for treatment of viral infection and method of use thereof.
The applicant listed for this patent is Ralf Geiben-Lynn. Invention is credited to Ralf Geiben-Lynn.
Application Number | 20140044776 14/058808 |
Document ID | / |
Family ID | 42826370 |
Filed Date | 2014-02-13 |
United States Patent
Application |
20140044776 |
Kind Code |
A1 |
Geiben-Lynn; Ralf |
February 13, 2014 |
SERPIN DRUGS FOR TREATMENT OF VIRAL INFECTION AND METHOD OF USE
THEREOF
Abstract
The invention includes a heparin activated Antithrombin III
encapsulated into a sterically stabilized anti-HLA-DR
immunoliposome for the treatment of a HIV infection in a human
patient.
Inventors: |
Geiben-Lynn; Ralf; (Concord,
NH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Geiben-Lynn; Ralf |
Concord |
NH |
US |
|
|
Family ID: |
42826370 |
Appl. No.: |
14/058808 |
Filed: |
October 21, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12727853 |
Mar 19, 2010 |
8563693 |
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14058808 |
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12391669 |
Feb 24, 2009 |
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12727853 |
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10892676 |
Jul 15, 2004 |
7510828 |
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12391669 |
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10057593 |
Jan 25, 2002 |
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10892676 |
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60264338 |
Jan 26, 2001 |
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Current U.S.
Class: |
424/450 ;
514/3.7 |
Current CPC
Class: |
A61K 9/127 20130101;
A61K 47/6911 20170801; A61K 31/727 20130101; A61P 31/14 20180101;
A61P 1/16 20180101; A61K 2300/00 20130101; A61P 31/22 20180101;
A61K 2300/00 20130101; A61P 31/20 20180101; A61K 31/727 20130101;
A61K 38/57 20130101; A61K 38/57 20130101; A61P 31/18 20180101 |
Class at
Publication: |
424/450 ;
514/3.7 |
International
Class: |
A61K 47/48 20060101
A61K047/48; A61K 9/127 20060101 A61K009/127 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made, in part, with Government support
under grant numbers N01 AI30048, N01 AI30049 and AI067854 awarded
by the National Institutes of Health. The Government has certain
rights in the invention.
Claims
1. A composition comprising heparin-antithrombin III and
liposomes.
2. The composition of claim 1, wherein said liposomes are
anti-HLA-DR immunoliposomes.
3. The composition of claim 1, further comprising a
pharmaceutically acceptable carrier or diluent.
4. The composition of claim 2, further comprising a
pharmaceutically acceptable carrier or diluent.
Description
RELATED APPLICATIONS
[0001] This application is a division of co-pending U.S. Ser. No.
12/727,853, filed Mar. 19, 2010, which in turn is a
continuation-in-part of U.S. Ser. No. 12/391,669, filed Feb. 24,
2009, which in turn is a continuation-in-part of U.S. Ser. No.
10/892,676, filed Jul. 15, 2004, now U.S. Pat. No. 7,510,828, which
in turn is a continuation of U.S. Ser. No. 10/057,593, filed Jan.
25, 2002, which in turn claims priority to U.S. Ser. No.
60/264,338, filed Jan. 26, 2001, all of which are incorporated
herein by reference in their entireties for all purposes.
FIELD OF THE INVENTION
[0003] The present invention relates to a method of antiviral
treatment using a serpin that inhibits serine protease and binds
heparin.
BACKGROUND OF THE INVENTION
[0004] The human retrovirus, human immunodeficiency virus (HIV)
causes Acquired Immunodeficiency Syndrome (AIDS), an incurable
disease in which the body's immune system breaks down leaving the
victim vulnerable to opportunistic infections, e.g., pneumonia, and
certain cancers, e.g., Kaposi's Sarcoma. AIDS is major global
health problem. The Joint United Nations Programme on HIV/AIDS
(UNAIDS) estimates that there are now over 34 million people living
with HIV or AIDS worldwide, some 28.1 million of those infected
individuals reside in impoverished sub-Saharan Africa. In the
United States, one out of every 250 people is infected with HIV or
have AIDS. Since the beginning of the epidemic, AIDS has killed
nearly 19 million people worldwide, including some 425,000
Americans. AIDS has replaced malaria and tuberculosis as the
world's deadliest infectious disease among adults and is the fourth
leading cause of death worldwide.
[0005] There is still no cure for AIDS. There is, however, an
armamentarium of antiretroviral drugs that prevent HIV from
reproducing and ravaging the body's immune system. One such class
of drugs are the reverse transcriptase inhibitors, e.g., abacavir,
delaviridine, didanosine, efavirenz, lamivudine, nevirapine,
stavudine, zalcitabine, and zidovudine, which attack an HIV enzyme
called reverse transcriptase. Another class of drugs is the
protease inhibitors, e.g., amprenavir, indinavir, nelfinavir,
ritonavir, and saquinavir, which inhibit HIV enzyme protease.
[0006] First introduced in 1995, these protease inhibitors are
widely used for the treatment of HIV infection alone or in
combination with other antiretroviral drugs. Today, approximately
215,000 of the estimated 350,000 patients receiving treatment for
HIV infection in the United States take at least one protease
inhibitor.
[0007] Highly active antiretroviral drug therapy (HAART) is a
widely used anti-HIV therapy that entails triple-drug protease
inhibitor-containing regimens that can completely suppress viral
replication (Stephenson, JAMA, 277: 614-6 (1997)). The persistence
of latent HIV in the body, however, has been underestimated. It is
now recognized that there exists a reservoir of HIV in perhaps tens
of thousands to a million long-lived resting "memory" T lymphocytes
(CD4), in which the HIV genome is integrated into the cells own DNA
(Stephenson, JAMA, 279: 641-2 (1998)). This pool of latently
infected cells is likely established during primary infection.
[0008] Such combination therapy is often only partially effective,
and it is unknown how much viral suppression is required to achieve
durable virologic, immunologic, and clinical benefit (Deeks, JAMA,
286: 224-6 (2001)). Anti-HIV drugs are highly toxic and can cause
serious side effects, including heart damage, kidney failure, and
osteoporosis. Long-term use of protease inhibitors has been linked
to peripheral wasting accompanied by abnormal deposits of body fat.
Other manifestations of metabolic disruptions associated with
protease inhibitors include increased levels of triglycerides and
cholesterol, pancreatitis, atherosclerosis, and insulin resistance
(Carr et al., LANCET, 351: 1881-3 (1998)). The efficacy of current
anti-HIV therapy is further limited by the complexity of regimens,
pill burden, and drug-drug interactions. Compliance with the toxic
effects of antiretroviral drugs make a lifetime of combination
therapy a difficult prospect and many patients cannot tolerate
long-term treatment with HAART. There is an urgent need for other
antiviral therapies due to poor adherence to combination therapy
regimes, which has led to the emergence of drug-resistant strains
of HIV. Other drugs may improve compliance by substantially
reducing the daily "pill burden" and simplifying the complicated
dietary guidelines associated with the use of current protease
inhibitors.
[0009] The HIV virus enters the body of an infected individual and
lives and replicates primarily in the white blood cells. The
hallmark of HIV infection, therefore, is a decrease in cells called
T-helper or CD4 cells of the immune system. The molecular mechanism
of HIV entry into cells involves specific interactions between the
viral envelope glycoproteins (env) and two target cell proteins,
CD4 and a chemokine receptor. HIV cell tropism is determined by the
specificity of the env for a particular chemokine receptor
(Steinberger et al., PROs. NATL. ACAD. ScI. USA. 97: 805-10
(2000)). T-cell-line-tropic (T-tropic) viruses (X4 viruses) require
the chemokine receptor CXR4 for entry. Macrophage (M)-tropic
viruses (R5 viruses) use CCR5 for entry (Berger et al., NATURE,
391: 240 (1998)). T-tropism is linked to various aspects of AIDS,
including AIDS dementia, and may be important in disseminating the
virus throughout the body and serving as a reservoir of virus in
the body.
[0010] CD8+ T-cells secrete soluble factor(s) capable of inhibiting
both R5- and X4-tropic strains of HIV and that are believed to play
a critical role in vivo in antiviral host defense (Garizino-Demo et
al., PROC. NATL. ACAD. SCI. USA, 96:111986-91 (1999)). These
inhibitory factors include CC-chemokines (Cocchi et al. SCIENCE,
270: 1811-5 (1995); Horuk et al., J. BIOL. CHEM., 273: 386-91
(1998); Pal et al., SCIENCE, 278: 695-8 (1997)), that bind to the
CCR5 coreceptor and inhibit R5 viral entry into cells
(Garizino-Demo et al., PROC. NATL. ACAD. SCI. USA., 96: 111986-91
(1999); Liu et al., CELL, 86: 367-77 (1996); Samson et al., NATURE,
382:722-5 (1996); Scarlatti et al., NAT. MED., 3: 1259-65 (1997))
as well as less well characterized soluble factor(s) produced by
CD8+ T-cells and termed CD8+ T-cell antiviral factor(s)
(hereinafter, CAF) capable of inhibiting both R5 and X4 HIV (Walker
et al., SCIENCE, 234: 1563-6 (1986); Chen et al., AIDS REs. Hum.
RETROVIRUSES, 9:1079-86 (1993); Mackewicz et al., PROC. NATL. ACAD.
SCI. USA, 92:2308-12 (1995); Mackewicz et al., J. GEN. VIROL., 81
Pt.S: 1261-4 (2000); Leith et al., AIDS, 11: 575-80 (1997); Le
Borgne et al., J. VIROL., 74: 4456-64 (2000); Tomaras et al., PROC.
NATL. ACAD. SCI. USA, 97: 3503-8 (2000)). These CC-chemokines,
however, do not account for all CAF antiviral activity released
from these cells, particularly since CAF can inhibit the
replication of X4 HIV strains that use CXCR4 and not CCR5 as a
coreceptor. The identity of the factor(s) released from CD8+
T-cells capable of inhibiting X4 HIV has remained elusive.
SUMMARY OF THE INVENTION
[0011] The invention provides compositions comprising substantially
purified serpin, which are useful in methods for the treatment and
prevention of HIV infection. The invention also includes methods
for the treatment and prevention of HIV infection comprising
contacting a composition of the invention with a human patient or
treating HIV infection by introducing into a cell susceptible to
HIV infection a DNA molecule encoding a serpin. Additionally, the
invention provides antibodies and kits useful in the detection,
treatment, and prevention of HIV infection.
[0012] The present invention provides a method of inhibiting the
infectivity of HIV by contacting an HIV virion with a composition
comprising a substantially purified preparation of a serpin, or
analog thereof. The composition is incubated with the virion for a
period of time sufficient to inhibit the infectivity of HIV. The
serpin may be selected from, but is not limited to a group
consisting of antithrombin (ATIII), protein C-inhibitor, activated
protein C, plasminogen activator inhibitor, and alpha-1-antitrypsin
A and may be pretreated chemically or enzymatically. e.g., elastase
pretreatment. The serpin may be either bovine-originated or
human-originated. In a preferred embodiment, the serpin, or analog
thereof, inhibits serine protease and binds heparin.
[0013] In a more preferred embodiment, a 43 kDa modified form of
antithrombin III (hereinafter, mATIII) from activated CD8+ T-cell
supernatants is used as an HIV inhibitory factor capable of
inhibiting the replication of both R5 and X4 HIV. In a most
preferred embodiment, the composition is comprised of 43 kDa ATIII
(hereinafter mATIII), R-ATIII, S-ATIII, or a combination
thereof.
[0014] The serpin composition may be used in a method of decreasing
the infectivity of HIV, if any is present, in a biological sample
by contacting the biological sample with an amount of serpin
sufficient to decrease the infectivity of HIV in the biological
sample. In a preferred embodiment, biological samples are contacted
with serpin at a concentration of at least about 2 U/ml final
biological sample volume. Biological samples which may treated for
HIV infection include, but are not limited to, blood, plasma,
serum, semen, cervical secretions, saliva, urine, breast milk, and
amniotic fluids.
[0015] The present invention also provides a method of treating HIV
infection by introducing a DNA molecule encoding a serpin into a
cell susceptible to HIV infection, and expressing the serpin in an
amount sufficient to inhibit infection of the cell by the HIV.
Similarly, the present invention provides a method of treating HIV
infection in a subject, the method comprising introducing into the
subject a producer cell that expresses a serpin in an amount
sufficient to inhibit infection of an endogenous cell of the
subject, the endogenous cell being susceptible to HIV infection. In
these methods, the expressed serpin preferably inhibits serine
protease and binds heparin. In a preferred embodiment, the
expressed serpin is ATIII, protein C-inhibitor, activated protein
C, plasminogen activator inhibitor, or .alpha.-1-antitrypsin. In a
more preferred embodiment, the expressed serpin is mATIII. R-ATIII,
S-ATIII, or combination thereof.
[0016] The present invention further provides a purification system
comprised of a serpin, or analog thereof, associated with a
surface, wherein the serpin is capable of inhibiting the
infectivity of HIV. A method of inhibiting the infectivity of HIV
is provided by the present invention where an HIV virion is
contacted with a composition having a surface that comprises
substantially purified serpin associated with the surface for a
length of time sufficient to inhibit the infectivity of HIV. In
particular, the serpin may be associated with a bead, chip, column,
or matrix. The present invention further provides a kit for
detecting a protein that inhibits the infectivity of HIV. In
particular, the kit comprises an antibody that specifically binds a
serpin, or analog thereof. Also, the detection reagent contained in
the kit is selected from the group consisting of an enzyme and a
radionucleotide. In these methods, the expressed serpin preferably
inhibits serine protease and binds heparin. In a preferred
embodiment, the expressed serpin is ATIII, protein C-inhibitor,
activated protein C, plasminogen activator inhibitor, or
alpha-1-antitrypsin. In a more preferred embodiment, the expressed
serpin is mATIII, R-ATIII, S-ATIII, or combination thereof.
[0017] The serpins of the invention can be used in a method of
decreasing the infectivity of HSV (i.e. HSV-1 or HSV-2) or HCV in a
biological sample containing cells susceptible to HSV infection by
identifying a biological sample in which a decrease or elimination
of HSV infectivity is desirable; and contacting the biological
sample containing cells susceptible to HSV infection with an
effective amount of S-antithromnbin or an amount of
S-antithrombin-bound-to-heparin, mATIII, R-ATIII, S-ATIII, or
combination thereof. In a preferred embodiment, biological samples
are contacted with serpin at a concentration of at least about 2
U/ml, 5 U/ml or 10 U/ml final biological sample volume. Biological
samples which may treated for HIV infection include, but are not
limited to, blood, plasma, serum, semen, cervical secretions,
saliva, urine, breast milk, and amniotic fluids.
[0018] These and other objects of the present invention will be
apparent from the detailed description of the invention provided
below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The present invention will be further understood from the
following description with reference to the figures in which:
[0020] FIGS. 1A-B detail members of the serpin protein superfamily.
This table was modified from: Irving et al., GENOME RESEARCH 10:
1845-64 (2000).
[0021] FIGS. 2A-C detail the analytical data used to identify
mATIII as a soluble HIV inhibitor secreted by CD8.sup.+ T-cells.
FIG. 2A is a C4 HPLC chromatogram of the substantially purified HIV
inhibitor, mATIII. FIG. 2B is a silver stained SDS-PAGE gel of the
substantially purified HIV inhibitor, mATIII. FIG. 2C is a table of
the partial protein sequence of HIV inhibitor (SEQ ID:1) obtained
by in-gel trypsin digestion of the SDS PAGE HIV inhibitor protein
band, elution of the resultant HIV-derived inhibitor peptides and
nano-electrospray tandem mass spectrometry.
[0022] FIGS. 3A-B demonstrate the antiviral effect of purified
bovine ATIII on HIV. FIG. 3A is a silver stained SDS-PAGE gel of
R-ATIII (porcine elastase treated; lane 1) and S-ATIII (undigested;
lane 2) used for the HIV inhibition tests. FIG. 3B is a graph
comparing the HIV inhibitory activity of varying concentrations of
R-ATIII and S-ATIII on X4 HIV and R5 HIV infectivity, respectively.
Virus inhibition was calculated using the buffer controls or the
enzyme controls.
[0023] FIGS. 4A-B are graphs comparing the effect of different
forms of ATIII on X4 HIV, SIV, and SHIV infectivity. FIG. 4A is a
graph comparing the effect of heat treatment (95.degree. C., 10 min
and 60.degree. C., 30 min treatment) on R- and S-ATIII-mediated X4
HIV inhibition using porcine elastase alone as an experimental
control. The inhibitory activity of a pre-latent ATIII (60.degree.
C., 24 h), and S-ATIII pretreated with V8 protease were also
tested. FIG. 4B is a graph comparing HIV inhibitory activity of
R-ATIII and S-ATIII on SIV and SHIV (SIV.sub.KU-1) infectivity.
Virus inhibition was calculated using the buffer controls or the
enzyme controls.
[0024] FIG. 5 is a graph showing in vivo protection of human
peripheral blood mononuclear cells (PBMC) during treatment with
Antithrombin III (ATIII) in NOD Cg-prkc-b2m.about./.about. mice.
10.sup.7 PBMC were infected with multidrug resistant HIV (100 ng
p24) and incubated at 37.degree. C. for 1 hour. 3.5.times.10.sup.6
in vitro infected PBMC were administered intra peritoneal into a
NOD) Cg-prkc-b2m-/- mice (n=5) and treated daily with 6 Units of
ATIII. Spleens were harvested at 14 days and number of cells were
counted under a light microscope. The data are expressed as mean
(.+-.standard errors of the means).
[0025] FIG. 6 is a graph showing suppression of HIV-1 expression in
TLR2 stimulated Tg cells. 5.times.10.sup.6/ml spleenocytes of Tg
mouse line 166 (Freitag et. al., 2001, J. Infect. Dis. 183,
1260-1268 (2001)), which contains multiple copies of the complete
proviral genome of HIV-1 strain NL4-5 were incubated with 10 ng/ml
bacterial lipoprotein (BLP) Pam3CSK4 (InvivoGen, San Diego,
Calif.), a TLR2 agonist, and different amounts of ATIII (0, 0.06,
0.3, 0.9 U/ml) for 48 hours (n=2). P24 HIV antigen was then assayed
by enzyme-linked immunosorbent assay (ELISA) in supernatants at 48
h using a commercial kit (p24 Coulter, Miami, Fla.).
[0026] FIG. 7 is a graph showing changes in gene expression
patterns of human PBMC after infection with HIV-1 but without
treatment of ATIII. 10.sup.5 human PBMC were acutely infected by
HIV-1 for 1 h at 37.degree. C., cells were washed and incubated for
40 h. Superarray's pathfinder Array.TM. (SABiosciences, Frederick,
Md.) was used to measure gene expression (n=3). Only significant
changes (p<0.05) are shown.
[0027] FIG. 8 is a graph showing changes in gene expression
patterns of human PBMC after treatment with ATIII but without
infection with HIV-1. 10.sup.5 human PBMC were treated with 2.4, 12
and 24 U ATIII/ml for 40 h. Superarray's pathfinder Array.TM.
(SABiosciences) was used to measure gene expression (n=3). Only
significant changes (p<0.05) are shown.
[0028] FIG. 9 is a graph showing changes in gene expression
patterns of human PBMC after infection with HIV-1 and treatment
with ATIII. 10.sup.5 human PBMC were acutely infected by HIV-1 for
1 h at 37.degree. C., cells were washed and treated with 2.4, 12
and 24 U ATIII/ml for 40 h. Superarray's pathfinder Array.TM.
(SABiosciences) was used to measure increase (A) or decrease in
gene expression (n=3). Only significant changes (p<0.05) are
shown.
[0029] FIG. 10 is a graph showing changes in gene expression
patterns HCV replicon after with ATIII. 10.sup.4 HCV replicon cells
were treated with 2.4, 7.2 and 24 U ATIII/ml for 40 h. Superarray's
pathfinder Array.TM. (SABiosciences) was used to measure gene
expression (n=3). Only significant changes (p<0.05) are
shown.
[0030] FIG. 11 is a graph showing the antiviral effect of ATIII and
heparin-ATIII complex on HCV.
[0031] FIG. 12 is a bar graph showing the reduction of viral load
in chronic infected rhesus macaques by sterically stabilized
anti-HLA-DR immunoliposomes encapsulating hep-ATIII. Two
Indian-origin macaques were chronically infected with
SIV.sub.mac251 for more than 450 days and then treated each via
s.c. route with 4 depots of a total of 1.5 ml of 0.05 mg/ml
hep-ATIII encapsulated in anti-HLA-DR immunoliposomes daily for two
days.
[0032] FIG. 13 is heat diagram of genes activated or down-regulated
by heparin activated, hep-ATIII, and ATIII in three rhesus
macaques.
DETAILED DESCRIPTION OF THE INVENTION
[0033] Definitions: As used herein, each of the following terms has
the meaning associated with it in this section.
[0034] The term "serpin," as used herein, is intended to include
native serpin polypeptide as well as any biologically active
fragment(s) or analog(s) thereof. The terms "fragment" and "analog"
are used interchangeably herein to describe serpins useful in the
methods of the present invention.
[0035] The term "substantially pure," as used herein, describes a
compound, e.g., a protein or polypeptide that has been separated
from components, which naturally accompany it. Typically, a
compound is substantially pure when at least 10%, more preferably
at least 20%, more preferably at least 50%, more preferably at
least 60%, more preferably at least 75%, more preferably at least
90%, and most preferably at least 99% of the total material (by
volume, by wet or dry weight, or by mole percent or mole fraction)
in a sample is the compound of interest. Purity can be measured by
any appropriate method, e.g., in the case of polypeptides by column
chromatography, gel electrophoresis or HPLC analysis. A compound,
e.g., a protein, is also substantially purified when it is
essentially free of naturally associated components or when it is
separated from the native contaminants which accompany it in its
natural state. Included within the meaning of the term
"substantially pure" as used herein is a compound, such as a
protein or polypeptide, which is homogeneously pure, for example,
where at least 95% of the total protein (by volume, by wet or dry
weight, or by mole percent or mole fraction) in a sample is the
protein or polypeptide of interest.
[0036] The term "specific binding" or "specifically binds," as used
herein, means a protein, such as an antibody, which recognizes and
binds a serpin, e.g. ATIII, or a ligand thereof, but does not
substantially recognize or bind other molecules in a sample.
[0037] The term "pharmaceutically acceptable carrier," as used
herein, means a chemical composition with which the active
ingredient may be combined and which, following the combination,
can be used to administer the active ingredient to a subject.
[0038] The term "physiologically acceptable" ester or salt, as used
herein, means an ester or salt form of the active ingredient which
is compatible with any other ingredients of the pharmaceutical
composition, which is not deleterious to the subject to which the
composition is to be administered.
[0039] The term "oily" liquid, as used herein, is one which
comprises a carbon-containing liquid molecule and which exhibits a
less polar character than water.
[0040] The term "additional ingredients," as used herein, include,
but are not limited to, one or more of the following: excipients;
surface active agents; dispersing agents; inert diluents:
granulating and disintegrating agents; binding agents; lubricating
agents; sweetening agents; flavoring agents; coloring agents;
preservatives; physiologically degradable compositions such as
gelatin; aqueous vehicles and solvents; oily vehicles and solvents;
suspending agents; dispersing or wetting agents; emulsifying
agents, demulcents; buffers; salts; thickening agents; fillers;
emulsifying agents; antioxidants; antibiotics; antifungal agents;
stabilizing agents; and pharmaceutically acceptable polymeric or
hydrophobic materials. Other "additional ingredients" which may be
included in the pharmaceutical compositions of the invention are
known in the art and described, for example, in Genaro, ed., 1985,
REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Co., Easton,
Pa., which is incorporated herein by reference.
[0041] One "unit" of ATIII enzymatic activity, as used herein, is
the activity present in 0.1 ml of normal human pooled plasma tested
in the presence of 0.1 unit of heparin (Damus and Rosenberg, METH.
ENZYMOL., 45. 653 (1976); PROTEOLYTIC ENZYMES: A PRACTICAL
APPROACH, eds. Beynon and Bond, p. 247 (1989)). One "unit" of
serpin enzymatic activity, as used herein, is understood to
represent the conventional measure of serpin activity as defined in
the art.
[0042] The term "transformation," as used herein, means introducing
DNA into a suitable host cell so that the DNA is replicable, either
as an extrachromosomal element, or by chromosomal integration.
[0043] The term "transfection," as used herein, refers to the
taking up of an expression vector by a suitable host cell, whether
or not any coding sequences are in fact expressed.
[0044] The term "infection," as used herein, refers to the
introduction of nucleic acids into a suitable host cell by use of a
virus or viral vector.
[0045] The term "antibody." as used herein, refers to an
immunoglobulin molecule that is able to specifically bind to a
specific epitope on an antigen.
I. HIV Inhibitors of the Present Invention
[0046] The present invention identifies antiretroviral activity of
serpins (FIG. 1). The invention also includes methods for the
treatment and prevention of HIV infection comprising contacting a
composition of the invention with a human patient or treating HIV
infection by introducing into a cell susceptible to HIV infection a
DNA molecule encoding a serpin. Additionally, the invention
includes antibodies and kits useful in the detection, treatment,
and prevention of HIV infection.
[0047] Serpins constitute a superfamily of structurally related
proteins found in eukaryotes, including humans (Wright, BIOASSAYS,
18: 453-64 (1996); Skinner et al., J. MOL. BIOL., 283: 9-14 (1998);
Huntington et al., J. MOL. BIOL., 293: 449-55 (1999); Interpro
#IPROO0215). Serpins are unusually large serine protease
inhibitors, e.g., ATIII, protein C-inhibitor, activated protein C,
plasminogen activator inhibitor, and alpha-1-antitrypsin. On a
molar basis, inhibitory serpins comprise some 10 percent of human
serum proteins.
[0048] While the Experimental Examples presented herein are
directed to antithrombin (hereinafter, ATIII), it is contemplated
that the present invention includes other serpins, as summarized in
FIG. 1, or peptide fragment(s) derived therefrom or analog(s)
thereof. In a preferred embodiment, the serpin binds heparin, and
inhibits both serine protease and HIV. The term "serpin"
encompasses naturally occurring serpins, as well as synthetic or
recombinant serpins. Further, the term "serpin" encompasses allelic
variants, species variants, and conservative amino acid
substitution variants. The term also encompasses full-length
serpins, as well as serpin fragments. It will thus be understood
that fragments of serpins variants, in amounts giving equivalent
biological activity to full-length serpins, can be used in the
methods of the invention, if desired. Fragments of serpin
incorporate at least the amino acid residues of serpins necessary
for a biological activity similar to that of intact serpin.
Examples of such fragments include the serpins presented in FIG.
1.
[0049] The term "serpin" also encompasses variants and functional
analogs of serpins having a homologous amino acid sequence with a
serpin. The present invention thus includes pharmaceutical
formulations comprising such serpin variants and functional
analogs, carrying modifications like substitutions, deletions,
insertions, inversions or cyclisations, but nevertheless having
substantially the biological activities of serpins.
[0050] According to the present invention, "homologous amino acid
sequence" means an amino acid sequence that differs by one or more
conservative amino acid substitutions, or by one or more
non-conservative amino acid substitutions, deletions, or additions
located at positions at which they do not destroy the biological
activities of the polypeptide. Conservative amino acid
substitutions typically include substitutions among amino acids of
the same class. These classes include, for example, (a) amino acids
having uncharged polar side chains, such as asparagine, glutamine,
serine, threonine, and tyrosine; (b) amino acids having basic side
chains, such as lysine, arginine, and histidine; (c) amino acids
having acidic side chains, such as aspartic acid and glutamic acid;
and (d) amino acids having nonpolar side chains, such as glycine,
alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan, and cysteine. Preferably, such a sequence
is at least 75%, preferably 80%, more preferably 85%, more
preferably 90%, and most preferably 95% homologous to the amino
acid sequence of the reference Serpin.
[0051] Serpin structure is typified by a multi-domain fold
containing a bundle of helices and a sandwich, and a welt-defined
C-terminal reactive region that acts as a `bait` for an appropriate
serine protease. Many serpins are high molecular weight (400 to 500
amino acids), extracellular, irreversible inhibitors of serine
proteases whose mechanism of inhibition involves dramatic
conformational changes (Skinner et al., J. MOL. BIOL., 283: 9-14
(1998); Huntington et al., J. MOL. BIOL., 293: 449-55 (1999)).
Significant tertiary structural changes may involve the insertion
of the reactive center peptide loop insert into a gap in a major
R-sheet forming a new strand (Stein and Carrell, NATURE STRUCT.
BIOL., 2: 96-113 (1995); Sharp et al., STRUCTURE, 7: 111-8 (1999)).
On the basis of strong sequence similarities, a number of proteins,
e.g., angiotensinogen, thyroxine binding globulin, and
corticosteroid binding globulin, with no known inhibitory activity,
are said to belong to this family (Stein and Carrell, NATURE
STRUCT. BIOL., 2: 96-113 (1995)).
[0052] Among the serpins, ATIII is a glycoprotein present in blood
plasma with a well defined role in blood clotting. Specifically,
ATIII is a potent inhibitor of the reactions of the coagulation
cascade with an apparent molecular weight between 54 kDa and 65 kDa
(Rosenberg and Damus. J. BIOL. CHEM., 248: 6490-505 (1973);
Nordenman et al., EUR. J. BIOCHEM., 78: 195-204 (1977); Kurachi et
al., BIOCHEMISTRY, 15: 373-7 (1976)) of which, some ten percent is
contributed by four glucosamine-base carbohydrate chains (Kurachi
et al., BIOCHEMISTRY, 15: 373-7 (1976); Petersen et al., IN THE
PHYSIOLOGICAL INHIBITORS OF COAGULATION AND FIBRINOLYSIS, (Collen,
Winman and Verstraete, eds) Elsevier. Amsterdam. p. 48 (1979)).
Although the name, ATIII, implies that it works only on thrombin,
it actually serves to inhibit virtually all of the coagulation
enzymes to at least some extent. The primary enzymes it inhibits
are factor Xa, factor IXa and thrombin (factor IIa). It also has
inhibitory actions on factor XIIa, factor XIa and the complex of
factor VIIa and tissue factor but not factor VIIa and activated
protein C. ATIII also inhibits trypsin, plasmin and kallikrein
(Charlotte and Church, SEMINARS IN HEMATOLOGY, 28:3-9 (1995). Its
ability to limit coagulation through multiple interactions makes it
one of the primary natural anticoagulant proteins.
[0053] ATIII acts as a relatively inefficient inhibitor on its own.
However, ATIII can be activated by a simple template mechanism, or
by an allosteric conformational change brought about by heparin
binding (Skinner et al., J. MOL. BIOL., 283: 9-14 (1998);
Huntington et al. J. MOL. BIOL., 293: 449-55 (1999); Belar et al.
J. BIOL. CHEM., 275: 8733-41 (2000)). When ATIII binds heparin, the
speed with which the reaction that causes inhibition occurs is
greatly accelerated; this makes the ATIII-heparin complex a vital
component of coagulation. This interaction is also the basis for
the use of heparin and low-molecular-weight heparins as medications
to produce anticoagulation.
[0054] There is a growing body of evidence that ATIII has
additional biological activity apart from its ability to inhibit
thrombin. For example, ATIII has been demonstrated as an
anti-inflammatory fraction in sepsis (Souter et at, CUT. CARE MED.,
29: 134-9 (2001)), as an anti-angiogenesis factor in tumor growth
(O'Reilly et at, SCIENCE, 285: 1926-8 (1999)), and is chemotactic
to neutrophils through the sydecan-4 receptor (Dunzendorfer et at,
BLOOD, 97: 1079-85 (2001); Kaneider et at, BIoCHEM, BwHYs. REs.
CoMMUN., 287: 42.6 (2001). The mechanism of action is so far not
entirely clear.
A. Purification and Identification of mATIII
[0055] Activated CD8.sup.+ T-cells produce at least two factors
capable of inhibiting the X4 strain HIV.sub.IIIB (Geiben-Lynn et
at, J. VIROL., 75: 8306-16 (2001)). These factors are distinct in
their size and ability to bind heparin. One of these factor binds
heparin at physiological salt concentration, elutes from a
purification column at 350 mM NaCl, and is retained by a 50 kDa cut
off Centricon filter. The other factor does not bind heparin at
physiological salt concentration and passes through a 50 kDa cut
off Centricon filter. The HIV inhibitory activity of these factors
is higher with bulk CD8.sup.+ T-cells of seropositive individuals
and HIV specific Cytotoxic T-Lymphocytes (CTL) compared to bulk
CD8.sup.+ T-cells of HIV seronegative individuals (Geiben-Lynn et
al., J. VIROL., 75: 8306-16 (2001)).
[0056] An X4 HIV inhibition assay (Geiben-Lynn et al., J. VIROL.,
75: 8306-16 (2001); Shapiro et al., FASEB J., 15: 115-22 (2001))
was used to purify the inhibitory activity found in the heparin
bound fraction of activated CD8+ T-cell supernatant (Geiben-Lynn et
al., J. VIROL., 75: 8306-16 (2001)). The HIV inhibitory activity
was purified to apparent homogeneity as measured by SDS-PAGE silver
staining and C4-HPLC (Van Patten et al., J. BIOL. CHEM., 274:
10268-76 (1999)) using heparin Sepharose and Superdex-200
size-exclusion-chromatography (FIG. 2A). The HIV inhibitory factor
was identified as a 43 kDa ATIII-like protein (mATIII; FIG. 2B), by
reverse-phase HPLC nano-electrospray tandem mass spectrometry
(.mu.LC/MS/MS) on a Finnigan LCQ quadrupole ion trap mass
spectrometer (FIG. 2C).
B. Characterization of the Antiretroviral Properties of ATIII
Forms
[0057] Analytical characterization of a CAF (Cf. FIG. 2) showed
that activated CD8+ T-cells modify ATIII to mATIII, a form with
enhanced ability to inhibit HIV infectivity. Therefore, the in
vitro antiretroviral activity of ATIII forms were measured and
compared (FIGS. 3 and 4). Under physiological conditions, ATIII
exists in different forms. In its most abundant configuration.
ATIII circulates in a quiescent form, L, form, in which its
reactive COOH-terminal loop is not fully exposed and cannot bind
target proteins. When bound to heparin, a stressed confirmation,
the S form of the molecule is induced: the reactive loop is
exposed, and thrombin-binding affinity is increased by up to a
factor of 100. The thrombin-ATIII complex then slowly dissociates,
and the reactive loop of ATIII is cleaved by the released thrombin.
The cleaved ATIII consists of disulfide-bonded A and B chains and
does not hind target proteases. Additionally, this cleavage induces
a conformational change to a relaxed confirmation, the R form, in
which the reactive loop is irreversibly inserted into an A-beta
sheet (Schreuder et al., NAT. STRUCT. BIOL., I: 48-54 (1994)).
[0058] An R-ATIII form was described as an anti-angiogenetic factor
capable of inhibiting tumor growth. This form of ATIII is cleaved
between Ser.sup.386 and Thr.sup.387 and can be generated by
digesting with porcine elastase (O'Reilly et al. SCIENCE, 285:
1926-8 (1999)). Other enzymes, which can cleave ATIII and produce
R-ATIII forms are thrombin (Arg.sup.394-Ser.sup.395), pancreatic
elastase (Val.sup.388-Iso.sup.389) and human neutrophil elastase
(Iso.sup.391-Ala.sup.392) (Evans et al. BIOCHEMISTRY, 31: 12629-42
(1992); Mourey et at., J. MoL. BIOL., 232: 223-41 (1993)). A
pre-latent ATIII, where the ATIII activity is still conserved and
the heparin binding affinity is retained, can be produced through
incubating S-ATIII at 60.degree. C. for 24 h under physiological
salt conditions (Larsson et al., J. BIOL. CHEM., 276: 11996-2002
(2001)).
[0059] To determine which form(s) of ATIII is capable of inhibiting
retrovirus infectivity, the R-ATIII, pre-latent ATIII and L-ATIII
were produced from a commercially available S-ATIII (serum purified
bovine S-ATIII; Sigma Chemical Co. St. Louis, Mo., USA; 0.2-0.4
U/.mu.g)). R-ATIII was obtained by incubating this S-ATIII (200
.mu.g/ml) for at 37.degree. C. in 20 mM Tris-HCl (pH 8.0)
containing 150 mM NaCl and 2.5 U/ml porcine pancreatic elastase
(Caibiochem Novabiochem Corporation, San Diego, Calif., USA; order
No. 324682. Essentially complete conversion of S-ATIII to R-ATIII
was obtained under these digestion conditions (FIG. 3A; O'Reilly et
al., SCIENCE, 285:1926-8 (1999)). In select studies, S-ATIII was
digested in PBS using an immobilized V-8 Protease Kit (PIERCE) for
1 h at 4.degree. C. according manufacturer's procedure.
[0060] X4 HTLV-IIIB (hereinafter X4 HIV; Chang et al., NATURE, 363:
466-9 (1993)), a prototypical T-tropic strain of HIV (American Type
Tissue Collection, Monassass, Va., USA: ATCC No. CRL-8543), was
used to assess the effect of ATIII on T-tropic HIV infection. The
quantity of virus in a specified suspension volume (e.g. 0.1 ml)
that will infect 50% of a number (n) of cell culture microplate
wells, or tubes, is termed the Tissue Culture Infectious Dose 50
[TCID.sub.50]. TCID.sub.50 is used as an alternative to determining
virus titre by plaqueing (which gives values as PFUs or
plaque-forming units). Karber, 1931.
[0061] Human T lymphoblastoid cells (H9 cells) expressing the human
leukocyte antigen proteins (HLA) 136, Bw62, and Cw3 were acutely
infected with X4 HIV at a MOI of 1.times.10.sup.-2 TCID.sub.50 per
milliliter. The infected H9 cells were resuspended to
5.times.10.sup.5 cells/ml in R20 cell culture medium. Two
milliliters of this suspension was pipetted into each well of a
24-well microtiter plate.
[0062] PM1 macrophage-like-cells were acutely infected with R5
HIV.sub.JR-CSF (hereinafter R5 HIV; Koyanagi, et al., SCIENCE, 236:
819-22, (1987)) to examine the ability of ATIII to affect
monocytropic HIV infection. The R5 HIV isolate, JR-CSF was
originally obtained from the cerebrospinal fluid of an HIV-infected
individual at autopsy. This strain shows properties characteristic
of a primary HIV isolates, e.g., it replicates efficiently in
primary blood cells but not in cell lines. That is, JR-CSF exhibits
properties more characteristics of clinical HIV isolates obtained
directly from the HIV patient. It is now a standard reference
strain representing macrophage tropic strains of HIV, PM1 cells
were acutely infected with HIV.sub.IIIB at a MOI of
1.times.10.sup.-2 TCID.sub.50 per milliliter.
[0063] Simian immunodeficiency virus (SIV) belongs to the family
Retroviridae (subfamily Lentivirinae) and is closely related to
human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2), the
etiologic agents of AIDS. Originally reported in 1985, the first
isolate from a rhesus macaque was called simian T-lymphotropic
virus III (STLV-III). The SIVmac239 viral strain (hereinafter
SIV.sub.239; P. Johnson, Harvard Medical School, Boston, Mass.,
USA) used in these studies is a dual-tropic infectious virus that
induces AIDS in rhesus macaque monkeys. SHIV.sub.KU-1 (Narayan and
Joag, AIDS Research and Reference Program, Division of AIDS, NIADS,
Bethesda, Md., USA) is a second dual-tropic strain of SIV used in
these studies. SHIV.sub.KU-1 is a biologically-pure suspension of
SHIV that is highly pathogenic in pigtailed macaques. The virus was
derived by sequential passage of the molecular construct of
SIV(mac)239XHIV-1-HxB2 through bone marrow of pigtailed macaque
monkeys (Joag et al, J. VIROLOGY, 70:3189-3197 (1996)).
[0064] The cell tropism of SIV in culture depends partially on the
strain of virus propagated and conditions of cell culture. In the
present studies, macaque T-cell line SEM-174 cells were acutely
infected with either SIV.sub.239 or SHIV.sub.KU-1 at a MOI of
1.times.10.sup.-2 TCID.sub.50 per milliliter.
[0065] As shown in FIG. 3B, R-ATIII inhibited X4 virus with
half-maximal inhibition (ID.sub.50) at approximately 25 .mu.g/ml.
The S-ATIII was more potent than R-ATIII and displayed, with an
ID.sub.50 at 10 .mu.g/ml (.about.3 U/ml), activity comparable to
the CD8.sup.+ T-cell modified form of ATIII (FIG. 38). This is
similar to the ID.sub.50 (5.5 .mu.g/ml), which was measured for the
mATIII and with 130 nM similar to that found for
Stromal-Derived-Factor (SDF-1), the only natural occurring ligand
found binding the CXCR4 coreceptor. (Geiben-Lynn et al., J. VIROL.,
75: 8306-16 (2001)).
[0066] As shown in FIG. 4, S-ATIII and R-ATIII-mediated antiviral
activity was resistant to inactivation by heat-treatment, as well
as pre-latent ATIII inhibited X4 HIV infectivity (FIG. 4A).
ATIII-mediated antiretroviral activity was not due to cytotoxicity
because ATIII treatment did not affect cell viability or cell
growth as judged by Trypan blue dye exclusion (data not shown). At
a concentration of 50 .mu.g/ml (15 U/ml), the S-ATIII inhibited SIV
and HSIV infectivity by 92 and 91%, respectively (FIG. 4B). R-ATIII
inhibited the simian retroviral strains to a lesser extent with 36
and 57% suppression of SIV core protein (p27), respectively (FIG.
4B).
[0067] This purified protein was of similar molecular size, but was
not the same as a previously described CD8.sup.+ T-cell antiviral
factor, CAF (Levy et al., IMMUNOL. TODAY, 17: 217-24 (1996)). That
is, the purified ATIII-like protein of the present invention is
similar in size to CAF and its ability to inhibit X4 viruses but
different in regard to heat stability (Geiben-Lynn et at, J.
VIROL., 75: 8306-16 (2001)). CAF has not been defined at a
molecular level, however, and it has only been tested as an
unfractionated supernatant. The anti-HIV activity of CAF may
reflect multiple factors involved with different points of
inhibition in the life cycle of HIV.
[0068] Purified CD8.sup.+ T-cell ATIII is a molecularly distinct
form of ATIII, Native, unmodified ATIII has a molecular weight of
54-65 kDa, whereas the purified ATIII form CD8.sup.+ T-cells was 43
kDa as judged by SDS-PAGE analysis. Purified CD8.sup.+ T-cell ATIII
is smaller than the S-ATIII and elutes from a heparin Sepharose
column at lower salt concentration (350 mM NaCl versus 1 M NaCl).
Purified CD8.sup.+ T-cell ATIII is also smaller that R-ATIII and
does not dissociate under reducing conditions used in the SDS-PAGE.
Finally, purified CD8.sup.+ T-cell ATIII and pre-latent ATIII
displayed similar anti-HIV potency in vitro but they differ in
molecular weight.
[0069] Under the conditions of enzyme pretreatment, V8 protease
preferentially digests the heparin-binding domain of ATIII.
Accordingly, the lack of antiretroviral activity in ATIII
preparation pretreated with V8 protease suggests that the
heparin-binding domain of ATIII is important for antiviral activity
(FIG. 4A). ATIII has been shown to bind to the syndecan family of
proteoglycans, which may mediate these biological activities. In
this regard, HIV. SIV, and SHIV have a requirement for syndecans
for attachment which facilitate HIV/SIV entry into cells
(Valenzuela-Fernandez et at, J. BIOL. CHEM., 276: 26550-8 (2001);
Saphire et at, J. VIROL., 75: 9187-200 (2001)). ATIII appears to
interact with the HIV-syndecan binding domain and that ATIII
inhibits HIV entry into cells, which might be synergistic with
other pathways. As such. ATIII and other serine protease inhibitors
offer the potential for improved efficacy and diminished toxicity
in the treatment of HIV and other viral diseases.
II. Use of the Present Invention for the Treatment, Prevention and
Detection of Retroviral Infection
[0070] The invention includes the use of a composition comprising
substantially purified ATIII. ATIII is capable of inhibiting the
infectivity of HIV as described herein, and thus is useful in
methods for the prevention of HIV infection in a patient or for
inhibiting the infectivity of HIV containing bodily fluids. The
ATIII to be used in the present invention is not particularly
limited as long as it has been purified to the extent that it can
be used as a pharmaceutical agent. For example, it can be purified
from whole blood, blood plasma, serum or serum obtained by
compression of coagulated blood. The starting material for
preparing ATIII may be, for example, fraction IV-1 or IV, or
supernatant I or II+III obtained by Cohn's fractionation of blood
plasma. ATIII can also be prepared by, for example, E. coli. cell
culture (e.g., EP-339919 to Isahiko et al.), genetic engineering
(e.g., EP-90505 to Botsuku and Roon), transgenic animal (Larrik and
Thomas, CURB. OPIN. BIOTECHNOL. 12: 41111-41118 (2001); Edmunds et
al., BLOOD 12: 4561-4571 (1998)), and the like. Alternatively, a
commercially available ATIII preparation can be used.
[0071] Compositions comprising substantially purified ATIII may
include ATIII alone, or in combination with other proteins. ATIII
may be substantially purified by any of the methods well known to
those skilled in the art. Substantially pure protein may be
purified by following known procedures for protein purification,
wherein an immunological, chromatographic, enzymatic, or other
assay is used to monitor purification at each stage in the
procedure. Protein purification methods are well known in the art,
and are described, for example in Deutscher et al., GUIDE TO
PROTEIN PURIFICATION, Harcourt Brace Iovanovich, San Diego (1990).
ATIII can be purified by a method described in, for example, U.S.
Pat. No. 3,842,061 to Anderson et al. and U.S. Pat. No. 4,340,589
to Uemura et al.
[0072] In one embodiment, the ATIII of the invention is a component
of a pharmaceutical composition, which may also comprise buffers,
salts, other proteins, and other ingredients acceptable as a
pharmaceutical composition. The invention also includes a modified
form of ATIII, which is capable of contacting HIV and inhibiting
the infectivity of HIV as described herein. The modified ATIII may
be used as a component of a composition for use in a method for
prevention of HIV infection of a patient or in the inhibition of
HIV infectivity of biological fluids.
[0073] The ATIII of the invention may be a molecule that comprises
the protein alone, or may include other components, such as protein
or other carbohydrate, or another molecule that may be covalently
linked to the ATIII, or may be non-covalently associated with the
ATIII.
[0074] The ATIII of the invention may be generated by enzymatic
digestion or chemical treatment of the full protein ATIII. Chemical
treatment methods may include, for example, digestion using mild
acid hydrolysis, treatment with 0.9 M guanidine (Carrell et al.,
NATURE, 353: 576-8 (1991)) or incubating S-ATIII in 0.25 mM
trisodium citrate at 60.degree. C. for 18 hours (Wardell et al.,
BIOCHEMISTRY, 36: 13133-42 (1997)). Enzymatic digestion methods may
include, for example, digestion using an elastase or other
protease. Enzymatic digestion methods may also include, for
example, digestion using a specific exoglycosidase (e.g.,
neuraminidase, mannosidase, fucosidase) or a specific
endoglycosidase (e.g., N-glycanase, O-glycanase).
[0075] In another embodiment, the ATIII of the invention may be
prepared using a biochemical synthesis method. Biochemical methods
for synthesizing proteins are well known to those skilled in the
art.
[0076] The ability to contact HIV virion may be assessed using
assays described herein in the Examples section. For example, the
virus may be incubated with the molecule comprising an ATIII of the
invention, placed over a sucrose cushion, and centrifuged. The
virus pellet obtained is resuspended, concentrated with
trichloroacetic acid (TCA) to concentrate the proteins, and
aliquots of the pellet and supernatant are analyzed by Western
blotting using antibodies to p24 (Nagashurmugam and Friedman, DNA
CELL BIOL. 15: 353-61 (1996)) or by an ELISA method.
[0077] In yet another embodiment, the molecule comprising the ATIII
of the invention is capable of inhibiting the infectivity of HIV in
a patient by contacting an HIV virion. The molecule comprising the
ATIII of the invention is included as a component in a
pharmaceutical composition that may be administered to a patient to
inhibit HIV infectivity or to prevent infection by HIV. The
inhibition of infectivity of HIV by the molecule comprising the
ATIII of the invention may be assessed as described herein. Such
methods may include p24 assay, reverse transcriptase activity
assay, or TCID.sub.50.
[0078] The invention also includes an antibody that is capable of
specifically binding to ATIII. The antibody of the invention may be
a monoclonal or a polyclonal antibody, or may be a synthetic,
humanized or phage displayed antibody. Antibodies can be intact
immunoglobulins derived from natural sources or from recombinant
sources and can be immunoreactive portions of intact
immunoglobulins. Antibodies are typically tetramers of
immunoglobulin molecules. The antibodies in the present invention
may exist in a variety of forms including, for example, polyclonal
antibodies, monoclonal antibodies, Fv, Fab and F(ab).sub.2, as well
as single chain antibodies and humanized antibodies (Harlow et al.,
1988, ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor, N.Y.;
Houston et al., PROC. NATL. ACAD. SCI. USA 85: 5879-83 (1988); Bird
et al., SCIENCE, 242: 423-6 (1988)). By the term "synthetic
antibody" as used herein, is meant an antibody which is generated
using recombinant DNA technology, such as, for example, an antibody
expressed by a bacteriophage as described herein. The term should
also be construed to mean an antibody which has been generated by
the synthesis of a DNA molecule encoding the antibody and which DNA
molecule expresses an antibody protein, or an amino acid sequence
specifying the antibody, wherein the DNA or amino acid sequence has
been obtained using synthetic DNA or amino acid sequence technology
which is available and well known in the art.
[0079] The invention also includes a kit for detecting a protein
that inhibits the infectivity of HIV. The proteins include ATIII.
The kit of the invention, may, for example, be an ELISA kit, which
includes an antibody, a detection reagent, and a reaction surface.
In one embodiment, the antibody is an antibody of the invention
that specifically binds with ATIII. The antibody may be any type of
antibody described herein and may be made using any of the methods
described herein. The reaction surface may be a microtiter plate,
such as an ELISA plate. The detection reagent may be any detection
reagent known to those skilled in the art. For example, the
detection reagent may be an enzyme, or a radionucleotide. In one
embodiment, the kit of the invention is an ELISA kit for detecting
the presence of ATIII in a bodily fluid such as serum of a human
patient.
[0080] The kit may include a microwell plate, an antibody that is
capable of specifically binding either ATIII, and a secondary
enzyme capable of binding the antibody of the invention and also
horseradish peroxidase. The ELISA kit of the invention may be used,
for example, to carry out an ELISA assay of a bodily fluid of a
patient, such as a serum sample. The assay may be used to detect
and quantify levels of ATIII present in the serum of the patient.
The quantity of ATIII in the patient's serum may be correlated with
the ability of the patient's serum to inhibit the infectivity of
HIV.
[0081] In another embodiment, the kit of the invention is a Western
Blotting or dot blotting kit for detecting the presence of ATIII in
a bodily fluid such as serum of a human patient.
[0082] The kits of the present invention may be used, for example,
to assess the susceptibility of a patient to HIV infection.
Patients with high susceptibility to HIV infection due to low
levels of ATIII may be treated with one of the pharmaceutical
compositions of the invention to enhance resistance of these
individuals to HIV infection. The correlation between the levels of
ATIII with the ability of a patient to inhibit the infectivity of
HIV is established using the procedures described in the
Experimental Examples presented herein.
[0083] The invention also includes a method of inhibiting the
infectivity of HIV in bodily fluids, or in infective oral
secretions. The method is useful in preventing HIV infection, or
inhibiting the infectivity of HIV. This method can be used, for
example to inhibit the infectivity of biological fluids, for
example in a hospital setting where medical personnel are exposed
to infectious HIV secretions.
[0084] In one embodiment, the method comprises contacting an HIV
virion with the human ATIII compositions described herein. In one
embodiment, the ATIII composition may comprise substantially
purified ATIII. The sample from a patient containing the HIV virion
may be obtained from any sample of bodily fluid, such as blood,
plasma, serum, semen, cervical secretions, saliva, urine, breast
milk, or amniotic fluids. In one embodiment, a composition
comprising substantially purified ATIII is contacted with an HIV
virion from a sample of a patient for a period of time sufficient
for the ATIII to inhibit the infectivity of HIV. The inhibition of
the infectivity of HIV can be assessed as described herein in the
Examples.
[0085] In another embodiment, the method of inhibiting the
infectivity of HIV comprises contacting an HIV virion obtained from
a bodily fluid sample of a patient with a composition having a
surface which contains a substantially purified human ATIII
associated with said surface. Examples of such surfaces include
plastic or other polymer surfaces, which are inert to reaction with
bodily fluids, and are considered biocompatible. In one embodiment
of the method of the invention, the composition having
substantially purified human ATIII associated with the surface is
contacted with a body fluid of a patient or an infective oral
secretion that contains an HIV virion. The composition is contacted
or incubated with the sample of bodily fluid containing the HIV
virion for a period of time sufficient to inhibit the infectivity
of HIV. The inhibition of the infectivity of HIV can be assessed as
described herein in the Examples section. For example, parameters
that are used to assess HIV replication, such as, for example, the
presence or absence of HIV specific components, such as nucleic
acid or protein, or in the latter case, the activity of HIV
specific components, such as reverse transcriptase, may be used to
assess inhibition of HIV in a sample.
[0086] The invention encompasses the preparation and use of
pharmaceutical compositions comprising a compound useful for the
prevention of HIV infection or inhibition of HIV infectivity as an
active ingredient. Such a pharmaceutical composition may consist of
the active ingredient alone, in a form suitable for administration
to a subject, or the pharmaceutical composition may comprise the
active ingredient and one or more pharmaceutically acceptable
carriers, one or more additional ingredients, or some combination
of these. The active ingredient may be present in the
pharmaceutical composition in the form of a physiologically
acceptable ester or salt, such as in combination with a
physiologically acceptable cation or anion, as is well known in the
art. Further, the ATIII (or biologically active analog thereof)
used in the present invention may contain pharmacologically
acceptable additives (e.g., carrier, excipient and diluent),
stabilizers or components necessary for formulating preparations,
which are generally used for pharmaceutical products, as long as it
does not adversely affect the object of the present invention.
[0087] Examples of the additives and stabilizers include
saccharides such as monosaccharides (e.g., glucose and fructose),
disaccharides (e.g., sucrose, lactose and maltose) and sugar
alcohols (e.g., mannitol and sorbitol); organic acids such as
citric acid, malic acid and tartaric acid and salts thereof (e.g.
sodium salt, potassium salt and calcium salt); amino acids such as
glycine, aspartic acid and glutamic acid and salts thereof (e.g.,
sodium salt); surfactants such as polyethylene glycol,
polyoxyethylene-polyoxypropylene copolymer and
polyoxyethylenesorbitan fatty acid ester; heparin; and albumin.
[0088] The formulations of the pharmaceutical compositions
described herein may be prepared by any method known or hereafter
developed in the art of pharmacology. In general, such preparatory
methods include the step of bringing the active ingredient into
association with a carrier or one or more other accessory
ingredients, and then, if necessary or desirable, shaping or
packaging the product into a desired single- or multi-dose
unit.
[0089] Although the descriptions of pharmaceutical compositions
provided herein are principally directed to pharmaceutical
compositions which are suitable for ethical administration to
humans, the skilled artisan will understand that such compositions
are generally suitable for administration to animals of all sorts.
Modification of pharmaceutical compositions suitable for
administration to humans in order to render the compositions
suitable for administration to various animals is well understood,
and the ordinarily skilled veterinary pharmacologist can design and
perform such modification with merely ordinary, if any,
experimentation. Subjects to which administration of the
pharmaceutical compositions of the invention is contemplated
include, but are not limited to, humans and other primates.
[0090] Pharmaceutical compositions that are useful in the methods
of the invention may be prepared, packaged, or sold in formulations
suitable for oral, rectal, vaginal, parenteral, topical, pulmonary,
intranasal, buccal, ophthalmic, or another route of administration.
The preferred mode is intravenous administration.
[0091] The ATIII and the above-mentioned ingredients are admixed as
appropriate to give powder, granule, tablet, capsule, syrup,
injection, and the like. Other contemplated formulations include
projected nanoparticles, liposomal preparations, resealed
erythrocytes containing the active ingredient, and immunologically
based formulations.
[0092] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in bulk, as a single unit dose, or as a
plurality of single unit doses. As used herein, a "unit dose" is
discrete amount of the pharmaceutical composition comprising a
predetermined amount of the active ingredient. The amount of the
active ingredient is generally equal to the dosage of the active
ingredient which would be administered to a subject or a convenient
fraction of such a dosage such as, for example, one-half or
one-third of such a dosage.
[0093] The relative amounts of the active ingredient, the
pharmaceutically acceptable carrier, and any additional ingredients
in a pharmaceutical composition of the invention will vary,
depending upon the identity, size, and condition of the subject
treated and further depending upon the route by which the
composition is to be administered. By way of example, the
composition may comprise between 0.1% and 100% (w/w) active
ingredient.
[0094] In addition to the active ingredient, a pharmaceutical
composition of the invention may further comprise one or more
additional pharmaceutically active agents. Particularly
contemplated additional agents include anti-emetics and scavengers
such as cyanide and cyanate scavengers. Controlled- or
sustained-release formulations of a pharmaceutical composition of
the invention may be made using conventional technology.
[0095] A formulation of a pharmaceutical composition of the
invention suitable for oral administration may be prepared,
packaged, or sold in the form of a discrete solid dose unit
including, but not limited to, a tablet, a hard or soft capsule, a
cachet, a troche, or a lozenge, each containing a predetermined
amount of the active ingredient. Other formulations suitable for
oral administration include, but are not limited to, a powdered or
granular formulation, an aqueous or oily suspension, an aqueous or
oily solution, or an emulsion.
[0096] A tablet comprising the active ingredient may, for example,
be made by compressing or molding the active ingredient, optionally
with one or more additional ingredients. Compressed tablets may be
prepared by compressing, in a suitable device, the active
ingredient in a free-flowing form such as a powder or granular
preparation, optionally mixed with one or more of a binder, a
lubricant, an excipient, a surface active agent, and a dispersing
agent. Molded tablets may be made by molding, in a suitable device,
a mixture of the active ingredient, a pharmaceutically acceptable
carrier, and at least sufficient liquid to moisten the mixture.
Pharmaceutically acceptable excipients used in the manufacture of
tablets include, but are not limited to, inert diluents,
granulating and disintegrating agents, binding agents, and
lubricating agents. Known dispersing agents include, but are not
limited to potato starch and sodium starch glycollate. Known
surface-active agents include, but are not limited to, sodium
lauryl sulphate. Known diluents include, but are not limited to,
calcium carbonate, sodium carbonate, lactose, microcrystalline
cellulose, calcium phosphate, calcium hydrogen phosphate, and
sodium phosphate. Known granulating and disintegrating agents
include, but are not limited to, corn starch and alginic acid.
Known binding agents include, but are not limited to, gelatin,
acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and
hydroxypropyl methylcellulose. Known lubricating agents include,
but are not limited to magnesium stearate, stearic acid, silica,
and talc.
[0097] Tablets may be non-coated or they may be coated using known
methods to achieve delayed disintegration in the gastrointestinal
tract of a subject, thereby providing sustained release and
absorption of the active ingredient. By way of example, a material
such as glyceryl monostearate or glyceryl distearate may be used to
coat tablets. Further by way of example, tablets may be coated
using methods described in U.S. Pat. Nos. 4,256,108 to Theeuwes;
4,160,452 to Theeuwes; and 4,265,874 to Bonsen et al., to form
osmotically controlled release tablets. Tablets may further
comprise a sweetening agent, a flavoring agent, a coloring agent, a
preservative, or some combination of these in order to provide
pharmaceutically elegant and palatable preparation.
[0098] Hard capsules comprising the active ingredient may be made
using a physiologically degradable composition, such as gelatin.
Such hard capsules comprise the active ingredient, and may further
comprise additional ingredients including, for example, an inert
solid diluent such as calcium carbonate, calcium phosphate, or
kaolin.
[0099] Soft gelatin capsules comprising the active ingredient may
be made using a physiologically degradable composition, such as
gelatin. Such soft capsules comprise the active ingredient, which
may be mixed with water or an oil medium such as peanut oil, liquid
paraffin, or olive oil.
[0100] Liquid formulations of a pharmaceutical composition of the
invention which are suitable for oral administration may be
prepared, packaged, and sold either in liquid form or in the form
of a dry product intended for reconstitution with water or another
suitable vehicle prior to use.
[0101] Liquid suspensions may be prepared using conventional
methods to achieve suspension of the active ingredient in an
aqueous or oily vehicle. Aqueous vehicles include, for example,
water and isotonic saline. Oily vehicles include, for example,
almond oil, oily esters, ethyl alcohol, vegetable oils such as
arachis, olive, sesame, or coconut oil, fractionated vegetable
oils, and mineral oils such as liquid paraffin. Liquid suspensions
may further comprise one or more additional ingredients including,
but not limited to, suspending agents, dispersing or wetting
agents, emulsifying agents, demulcents, preservatives, buffers,
salts, flavorings, coloring agents, and sweetening agents. Oily
suspensions may further comprise a thickening agent. Known
suspending agents include, but are not limited to, sorbitol syrup,
hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone,
gum tragacanth, gum acacia, and cellulose derivatives such as
sodium carboxymethylcellulose, methylcellulose,
hydroxypropylmethylcellulose. Known dispersing or wetting agents
include, but are not limited to, naturally-occurring phosphatides
such as lecithin, condensation products of an alkylene oxide with a
fatty acid, with a long chain aliphatic alcohol, with a partial
ester derived from a fatty acid and a hexitol, or with a partial
ester derived from a fatty acid and a hexitol anhydride (e.g.,
polyoxyethylene stearate, heptadecaethyleneoxycetanol,
polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan
monooleate, respectively). Known emulsifying agents include, but
are not limited to, lecithin and acacia. Known preservatives
include, but are not limited to methyl, ethyl, or
n-propyl-para-hydroxybenzoates, ascorbic acid, and sorbic acid.
Known sweetening agents include, for example, glycerol, propylene
glycol, sorbitol, sucrose, and saccharin. Known thickening agents
for oily suspensions include, for example, beeswax, hard paraffin,
and acetyl alcohol.
[0102] Liquid solutions of the active ingredient in aqueous or oily
solvents may be prepared in substantially the same manner as liquid
suspensions, the primary difference being that the active
ingredient is dissolved, rather than suspended in the solvent.
Liquid solutions of the pharmaceutical composition of the invention
may comprise each of the components described with regard to liquid
suspensions, it being understood that suspending agents will not
necessarily aid dissolution of the active ingredient in the
solvent. Aqueous solvents include, for example, water and isotonic
saline. Oily solvents include, for example, almond oil, oily
esters, ethyl alcohol, vegetable oils such as arachis, olive,
sesame, or coconut oil, fractionated vegetable oils, and mineral
oils such as liquid paraffin.
[0103] Powdered and granular formulations of a pharmaceutical
preparation of the invention may be prepared using known methods.
Such formulations may be administered directly to a subject, used,
for example, to form tablets, to fill capsules, or to prepare an
aqueous or oily suspension or solution by addition of an aqueous or
oily vehicle thereto. Each of these formulations may further
comprise one or more of dispersing or wetting agent, a suspending
agent, and a preservative. Additional excipients, such as fillers
and sweetening, flavoring, or coloring agents, may also be included
in these formulations.
[0104] A pharmaceutical composition of the invention may also be
prepared, packaged, or sold in the form of oil-in-water emulsion or
a water-in-oil emulsion. The oily phase may be a vegetable oil such
as olive or arachis oil, a mineral oil such as liquid paraffin, or
a combination of these. Such compositions may further comprise one
or more emulsifying agents such as naturally occurring gums such as
gum acacia or gum tragacanth, naturally-occurring phosphatides such
as soybean or lecithin phosphatide, esters or partial esters
derived from combinations of fatty acids and hexitol anhydrides
such as sorbitan monooleate, and condensation products of such
partial esters with ethylene oxide such as polyoxyethylene sorbitan
monooleate. These emulsions may also contain additional ingredients
including, for example, sweetening or flavoring agents.
[0105] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in a formulation suitable for rectal
administration. Such a composition may be in the form of, for
example, a suppository, a retention enema preparation, and a
solution for rectal or colonic irrigation.
[0106] Suppository formulations may be made by combining the active
ingredient with a non-irritating pharmaceutically acceptable
excipient which is solid at ordinary room temperature (i.e. about
20.degree. C.) and which is liquid at the rectal temperature of the
subject (i.e., about 37.degree. C. in a healthy human). Suitable
pharmaceutically acceptable excipients include, but are not limited
to, cocoa butter, polyethylene glycols, and various glycerides.
Suppository formulations may further comprise various additional
ingredients including, but not limited to, antioxidants and
preservatives.
[0107] Retention enema preparations or solutions for rectal or
colonic irrigation may be made by combining the active ingredient
with a pharmaceutically acceptable liquid carrier. As is well known
in the art, enema preparations may be administered using, and may
be packaged within, a delivery device adapted to the rectal anatomy
of the subject. Enema preparations may further comprise various
additional ingredients including, but not limited to, antioxidants
and preservatives.
[0108] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in a formulation suitable for vaginal
administration. Such a composition may be in the form of, for
example, a suppository, an impregnated or coated
vaginally-insertable material such as a tampon, a douche
preparation, a gel or cream or solution for vaginal irrigation.
[0109] Methods for impregnating or coating a material with a
chemical composition are known in the art, and include, but are not
limited to methods of depositing or binding a chemical composition
onto a surface, methods of incorporating a chemical composition
into the structure of a material during the synthesis of the
material (i.e., such as with a physiologically degradable
material), and methods of absorbing an aqueous or oily solution or
suspension into an absorbent material, with or without subsequent
drying.
[0110] Douche preparations or solutions for vaginal irrigation may
be made by combining the active ingredient with a pharmaceutically
acceptable liquid carrier. As is well known in the art, douche
preparations may be administered using, and may be packaged within,
a delivery device adapted to the vaginal anatomy of the
subject.
[0111] Douche preparations may further comprise various additional
ingredients including, but not limited to, antioxidants,
antibiotics, antifungal agents, and preservatives.
[0112] Additional delivery methods for administration of compounds
include a drug delivery device, such as that described in U.S. Pat.
No. 5,928,195 to Malamud et al.
[0113] As used herein, "parenteral administration" of a
pharmaceutical composition includes any route of administration
characterized by physical breaching of a tissue of a subject and
administration of the pharmaceutical composition through the breach
in the tissue. Parenteral administration thus includes, but is not
limited to, administration of a pharmaceutical composition by
injection of the composition, by application of the composition
through a surgical incision, by application of the composition
through a tissue-penetrating non-surgical wound, and the like. In
particular, parenteral administration is contemplated to include,
but is not limited to, subcutaneous, intraperitoneal,
intramuscular, intrasternal injection, and kidney dialytic infusion
techniques.
[0114] Formulations of a pharmaceutical composition suitable for
parenteral administration comprise the active ingredient combined
with a pharmaceutically acceptable carrier, such as sterile water
or sterile isotonic saline. Such formulations may be prepared,
packaged, or sold in a form suitable for bolus administration or
for continuous administration. Injectable formulations may be
prepared, packaged, or sold in unit dosage form, such as in
ampoules or in multi-dose containers containing a preservative.
Formulations for parenteral administration include, but are not
limited to, suspensions, solutions, emulsions in oily or aqueous
vehicles, pastes, and implantable sustained-release or
biodegradable formulations. Such formulations may further comprise
one or more additional ingredients including, but not limited to,
suspending, stabilizing, or dispersing agents. In one embodiment of
a formulation for parenteral administration, the active ingredient
is provided in dry (i.e., powder or granular) form for
reconstitution with a suitable vehicle (e.g., sterile pyrogen-free
water) prior to parenteral administration of the reconstituted
composition.
[0115] The pharmaceutical compositions may be prepared, packaged,
or sold in the form of a sterile injectable aqueous or oily
suspension or solution. This suspension or solution may be
formulated according to the known art, and may comprise, in
addition to the active ingredient, additional ingredients such as
the dispersing agents, wetting agents, or suspending agents
described herein. Such sterile injectable formulations may be
prepared using a non-toxic parenterally-acceptable diluent or
solvent, such as water or 1,3-butane diol, for example. Other
acceptable diluents and solvents include, but are not limited to,
Ringer's solution, isotonic sodium chloride solution, and fixed
oils such as synthetic mono- or di-glycerides. Other
parentally-administrable formulations that are useful include those
which comprise the active ingredient in microcrystalline form, in a
liposomal preparation, or as a component of a biodegradable polymer
systems. Compositions for sustained release or implantation may
comprise pharmaceutically acceptable polymeric or hydrophobic
materials such as an emulsion, an ion exchange resin, a sparingly
soluble polymer, or a sparingly soluble salt.
[0116] Formulations suitable for topical administration include,
but are not limited to, liquid or, semi-liquid preparations such as
liniments, lotions, oil-in-water or water-in-oil emulsions such as
creams, ointments or pastes, and solutions or suspensions.
Topically-administrable formulations may, for example, comprise
from about 1% to about 10% (w/w) active ingredient, although the
concentration of the active ingredient may be as high as the
solubility limit of the active ingredient in the solvent.
Formulations for topical administration may further comprise one or
more of the additional ingredients described herein.
[0117] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in a formulation suitable for pulmonary
administration via the buccal cavity. Such a formulation may
comprise dry particles which comprise the active ingredient and
which have a diameter in the range from about 0.5 to about 7
nanometers, and preferably from about 1 to about 6 nanometers. Such
compositions are conveniently in the form of dry powders for
administration using a device comprising a dry powder reservoir to
which a stream of propellant may be directed to disperse the powder
or using a self-propelling solvent/powder-dispensing container such
as a device comprising the active ingredient dissolved or suspended
in a low-boiling propellant in a scaled container. Preferably, such
powders comprise particles wherein at least 98% of the particles by
weight have a diameter greater than 0.5 nanometers and at least 95%
of the particles by number have a diameter less than 7 nanometers.
More preferably, at least 95% of the particles by weight have a
diameter greater than 1 nanometer and at least 90% of the particles
by number have a diameter less than 6 nanometers. Dry powder
compositions preferably include a solid fine powder diluent such as
sugar and are conveniently provided in a unit dose form.
[0118] Low boiling propellants generally include liquid propellants
having a boiling point of below 65.degree. F. at atmospheric
pressure. Generally the propellant may constitute 50 to 99.9% (w/w)
of the composition, and the active ingredient may constitute 0.1 to
20% (w/w) of the composition. The propellant may further comprise
additional ingredients such as a liquid non-ionic or solid anionic
surfactant or a solid diluent (preferably having a particle size of
the same order as particles comprising the active ingredient).
[0119] Pharmaceutical compositions of the invention formulated for
pulmonary delivery may also provide the active ingredient in the
form of droplets of a solution or suspension. Such formulations may
be prepared, packaged, or sold as aqueous or dilute alcoholic
solutions or suspensions, optionally sterile, comprising the active
ingredient, and may conveniently be administered using any
nebulization or atomization device. Such formulations may further
comprise one or more additional ingredients including, but not
limited to, a flavoring agent such as saccharin sodium, a volatile
oil, a buffering agent, a surface active agent, or a preservative
such as methylhydroxybenzoate. The droplets provided by this route
of administration preferably have an average diameter in the range
from about 0.1 to about 200 nanometers.
[0120] The formulations described herein as being useful for
pulmonary delivery are also useful for intranasal delivery of a
pharmaceutical composition of the invention.
[0121] Another formulation suitable for intranasal administration
is a coarse powder comprising the active ingredient and having an
average particle from about 0.2 to 500 micrometers. Such a
formulation is administered in the manner in which snuff is taken
i.e., by rapid inhalation through the nasal passage from a
container of the powder held close to the nares.
[0122] Formulations suitable for nasal administration may, for
example, comprise from about as little as 0.1% (w/w) and as much as
100% (w/w) of the active ingredient, and may further comprise one
or more of the additional ingredients described herein.
[0123] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in a formulation suitable for buccal
administration. Such formulations may, for example, be in the form
of tablets or lozenges made using conventional methods, and may,
for example, 0.1 to 20% (w/w) active ingredient, the balance
comprising an orally dissolvable or degradable composition and,
optionally, one or more of the additional ingredients described
herein. Alternately, formulations suitable for buccal
administration may comprise a powder or an aerosolized or atomized
solution or suspension comprising the active ingredient. Such
powdered, aerosolized, or aerosolized formulations, when dispersed,
preferably have an average particle or droplet size in the range
from about 0.1 to about 200 nanometers, and may further comprise
one or more of the additional ingredients described herein.
[0124] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in a formulation suitable for
ophthalmic administration. Such formulations may, for example, be
in the form of eye drops including, for example, a 0.1-1.0% (w/w)
solution or suspension of the active ingredient in an aqueous or
oily liquid carrier. Such drops may further comprise buffering
agents, salts, or one or more other of the additional ingredients
described herein. Other ophthalmalmically-administrable
formulations that are useful include those which comprise the
active ingredient in microcrystalline form or in a liposomal
preparation.
[0125] The mixture of ATIII and pharmacologically acceptable
additives is preferably prepared as a lyophilized product, and
dissolved when in use. Such preparation can be prepared into a
solution containing about 1-100 units/ml of ATIII, by dissolving it
in distilled water for infection or sterile purified water. More
preferably, it is adjusted to have a physiologically isotonic salt
concentration and a physiologically desirable pH value (pH
6-8).
[0126] ATIII has been shown to be well-tolerated when administered
at a dose of .about.100 U/kg/day (Warren et al., JAMA 286: 1869-78
(2001)) and has an overall elimination half-life with 18.6 h was
demonstrated (Ilias et al., INTENSIVE CARE MEDICINE 26: 7104-7115
(2000)). While the dose is appropriately determined depending on
symptom, body weight, sex, animal species and the like, it is
generally 1-1,000 units/kg body weight/day, preferably 10-500
units/kg body weight/day of ATIII for a human adult, which is
administered in one to several doses a day. In the case of
intravenous administration, for example, the dose is preferably
10-100 units/kg body weight/day. The compound may be administered
as frequently as several times daily, or it may be administered
less frequently, such as once a day, once a week, once every two
weeks, once a month, or even less frequently, such as once every
several months or even once a year or less. The frequency of the
dose will be readily apparent to the skilled artisan and will
depend upon any number of factors, such as, but not limited to, the
type and severity of the disease being treated, the type and age of
the animal, etc.
[0127] The present invention further provides host cells
genetically engineered to contain the polynucleotides encoding
ATIII or analogs of ATIII and expressing the ATIII polypeptide in
an amount sufficient to inhibit infection of the cell by HIV. Still
further, the present invention provides a method of treating HIV
infection in a subject, introducing into the subject a producer
cell that expresses ATIII in a sufficient amount to inhibit
infection of an endogenous cell of the subject. For example, such
host cells may contain nucleic acids encoding ATIII and introduced
into the host cell using known transformation, transfection or
infection methods. The present invention still further provides
host cells genetically engineered to express the polynucleotides of
ATIII, wherein such polynucleotides are in operative association
with a regulatory sequence heterologous to the host cell, which
drives expression of the polynucleotides in the cell. See, for
example, U.S. Pat. No. 4,632,981 to Bock and Lawn; and EP-90505 to
Botsuku and Roon.
[0128] Knowledge of ATIII nucleic acid sequences allows for
modification of cells to permit, or increase, expression of
endogenous polypeptide. Cells can be modified (e.g., by homologous
recombination) to provide increased polypeptide expression by
replacing, in whole or in part, the naturally occurring promoter
with all or part of a heterologous promoter so that the cells
express the polypeptide at higher levels. The heterologous promoter
is inserted in such a manner that it is operatively linked to the
encoding sequences. See, for example, PCT International Publication
No. WO94/12650 by Hartlein et al., PCT International Publication
No. WO 92/20808 by Smithies, and PCT International Publication No.
WO 91/09955 by Chappel. It is also contemplated that, in addition
to heterologous promoter DNA, amplifiable marker DNA (e.g., ada,
dhfr, and the multifunctional CAD gene which encodes carbamyl
phosphate synthase, aspartate transcarbamylase, and dihydroorotase)
and/or intron DNA may be inserted along with the heterologous
promoter DNA. If linked to the coding sequence, amplification of
the marker DNA by standard selection methods results in
co-amplification of the desired protein coding sequences in the
cells.
[0129] The host cell can be a higher eukaryotic host cell, such as
a mammalian cell, a lower eukaryotic host cell, such as a yeast
cell, or the host cell can be a prokaryotic cell, such as a
bacterial cell. Introduction of the recombinant construct into the
host cell can be effected by calcium phosphate transfection, DEAE,
dextran mediated transfection, or electroporation Davis et al.,
BASIC METHODS IN MOLECULAR BIOLOGY (1986)). The host cells
containing a polynucleotide encoding ATIII, can be used in
conventional manners to produce the gene product encoded by the
isolated analog or fragment (in the case of an open reading frame)
or can be used to produce a heterologous protein under the control
of the EMF.
[0130] Any host/vector system can be used to express one or more
ATIII protein forms. Potential hosts include, but are not limited
to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, 293
cells, and Sf9 cells, as well as prokaryotic host such as E. coli
and B. subtilis. The most preferred cells are those which do not
normally express the particular polypeptide or protein or which
expresses the polypeptide or protein at low natural level. Mature
proteins can be expressed in mammalian cells, yeast, bacteria, or
other cells under the control of appropriate promoters. Cell-free
translation systems can also be employed to produce such proteins
using RNAs derived from the DNA constructs of the present
invention. Appropriate cloning and expression vectors for use with
prokaryotic and eukaryotic hosts are described by Sambrook et al.,
in MOLECULAR CLONING: A LABORATORY MANUAL, SECOND EDITION, COLD
SPRING HARBOR, NEW YORK (1989), the disclosure of which is hereby
incorporated by reference.
[0131] Various mammalian cell culture systems can also be employed
to express recombinant ATIII protein. Examples of mammalian
expression systems include the COS-7 lines of monkey kidney
fibroblasts, described by Gluzman, CELL, 23:175-82 (1981). Other
cell lines capable of expressing a compatible vector are, for
example, the C127, monkey COS cells, Chinese Hamster Ovary (CHO)
cells, human kidney 293 cells, human epidermal A431 cells, human
Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate
cell lines, normal diploid cells, cell strains derived from in
vitro culture of primary tissue, primary explants, HeLa cells,
mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells. Mammalian
expression vectors will comprise an origin of replication, a
suitable promoter and also any necessary ribosome binding sites,
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking
nontranscribed sequences. DNA sequences derived from the SV40 viral
genome, for example, SV40 origin, early promoter, enhancer, splice,
and polyadenylation sites may be used to provide the required
nontranscribed genetic elements. Recombinant polypeptides and
proteins produced in bacterial culture are usually isolated by
initial extraction from cell pellets, followed by one or more
salting-out, aqueous ion exchange or size exclusion chromatography
steps. Protein refolding steps can be used, as necessary, in
completing configuration of the mature protein. Finally, high
performance liquid chromatography (HPLC) can be employed for final
purification steps. Microbial cells employed in expression of
proteins can be disrupted by any convenient method, including
freeze-thaw cycling, sonication, mechanical disruption, or use of
cell lysing agents.
[0132] The present invention also provides a method of treating HIV
infection where a DNA encoding a serpin, e.g., ATIII, or analog
thereof, is introduced into a cell susceptible to HIV infection and
expressed in a sufficient amount to inhibit infection of the cell
by the HIV. That is, the invention provides gene therapy to treat
retrovirus-induced disease states involving serpin, e.g., ATIII.
Delivery of a functional gene encoding serpin to appropriate cells
is effected ex vivo, in situ, or in vivo by use of vectors, and
more particularly viral vectors (e.g. adenovirus, adeno-associated
virus, or a retrovirus), or ex vivo by use of physical DNA transfer
methods (e.g., liposomes or chemical treatments). See, for example,
Anderson, NATURE, 392(6679Suppl.): 2530 (1998); see also,
Friedmann, SCIENCE, 244: 1275-81 (1989); Verma, Sa. Am., 263: 68-84
(1990); Miller, NATURE, 357: 455-60 (1992). Introduction of a
serpin gene encoding the can also be accomplished with
extrachromosomal substrates (transient expression) or artificial
chromosomes (stable expression). Cells may also be cultured ex vivo
in the presence of serpin in order to proliferate or to produce a
desired effect on or activity in such cells. Treated cells can then
be introduced in vivo for therapeutic purposes. Alternatively, it
is contemplated that antisense therapy or gene therapy could be
applied to negatively regulate the expression of serpins of the
invention.
[0133] Other methods inhibiting expression of a protein include the
introduction of antisense molecules to the nucleic acids of the
present invention, their complements, or their translated RNA
sequences, by methods known in the art. Further, the serpins can be
inhibited by using targeted deletion methods, or the insertion of a
negative regulatory element such as a silencer, which is tissue
specific.
[0134] The present invention still further provides cells
genetically engineered in vivo to express polynucleotides encoding
serpin, e.g., ATIII, wherein such polynucleotides are in operative
association with a regulatory sequence heterologous to the host
cell which drives expression of the polynucleotides in the cell.
These methods can be used to increase or decrease the expression of
the serpin polynucleotides.
[0135] In another embodiment of the present invention, cells and
tissues may be engineered Ito express an endogenous gene comprising
serpin, e.g., ATIII, under the control of inducible regulatory
elements, in which case the regulatory sequences of the endogenous
gene may be replaced by homologous recombination. As described
herein, gene targeting can be used to replace a gene's existing
regulatory region with a regulatory sequence isolated from a
different gene or a novel regulatory sequence synthesized by
genetic engineering methods. Such regulatory sequences may be
comprised of promoters, enhancers, scaffold-attachment regions,
negative regulatory elements, transcriptional initiation sites,
regulatory protein binding sites or combinations of said sequences.
Alternatively, sequences which affect the structure or stability of
the RNA or protein produced may be replaced, removed, added, or
otherwise modified by targeting. These sequence include
polyadenylation signals, mRNA stability elements, splice sites,
leader sequences for enhancing or modifying transport or secretion
properties of the protein, or other sequences which alter or
improve the function or stability of protein or RNA molecules.
[0136] In all the above embodiments involving augmentation of
cellular serpin. e.g., ATIII, expression, the targeting event may
be a simple insertion of the regulatory sequence, placing the gene
under the control of the new regulatory sequence, e.g., inserting a
new promoter or enhancer or both upstream of a gene. Alternatively,
the targeting event may be a simple deletion of a regulatory
element, such as the deletion of a tissue-specific negative
regulatory element. Alternatively, the targeting event may replace
an existing element; for example, a tissue-specific enhancer can be
replaced by an enhancer that has broader or different cell-type
specificity than the naturally occurring elements. Here, the
naturally occurring sequences are deleted and new sequences are
added. In all cases, the identification of the targeting event may
be facilitated by the use of one or more selectable marker genes
that are contiguous with the targeting DNA, allowing for the
selection of cells in which the exogenous DNA has integrated into
the host cell genome. The identification of the targeting event may
also be facilitated by the use of one or more marker genes
exhibiting the property of negative selection, such that the
negatively selectable marker is linked to the exogenous DNA, but
configured such that the negatively selectable marker flanks the
targeting sequence, and such that a correct homologous
recombination event with sequences in the host cell genome does not
result in the stable integration of the negatively selectable
marker. Markers useful for this purpose include the Herpes Simplex
Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine
phosphoribosyl-transferase (gpt) gene.
[0137] The gene targeting or gene activation techniques which can
be used in accordance with this aspect of the invention are more
particularly described in U.S. Pat. No. 5,272,071 to Chappel; U.S.
Pat. No. 5,578,461 to Sherwin et al.; international Application No
PCT/US92/09627 (WO93/09222) by Selden et al.; and International
Application No. PCT/US90/06436 (WO91/06667) by Skoultchi et al.,
each of which is incorporated by reference herein in its
entirety.
[0138] The present invention is described in more detail in the
following by illustrative Examples, to which the present invention
is not limited.
EXAMPLES
[0139] These Examples are provided for the purpose of illustration
only and the invention should in no way be construed as being
limited to these Examples, but rather should be construed to
encompass any and all variations which become evident as a result
of the teaching provided herein.
Example 1
HPLC Purification of an HIV Inhibitory Factor from CD8.sup.+
T-Cells and its Identification as an Antithrombin III Using
Nano-Electrospray Tandem Mass Spectrometry
[0140] To purify the ATIII-like HIV inhibitory factor, HIV-specific
CTL or bulk CD8.sup.+ T-cells of long-term non-progressors were
cultivated in vitro and stimulated with CD3 crosslinking
(Geiben-Lynn et al., J. VIROL., 75: 8306-16 (2001)) in either 10%
heat-inactivated fetal bovine serum or 10% heat-inactivated human
serum. After 4 h at 37.degree. C., media was collected,
centrifuged, and applied to a heparin Sepharose column. The column
was eluted with a continuous gradient to 1 M NaCl in
phosphate-buffered saline (PBS, pH 7.4). Inhibitory fractions were
pooled, and concentrated with a Centricon 50K centrifugal
concentrator. The sample was applied to a Superdex 200 column.
Fractions that inhibited were applied to a Vydac RP-4 HPLC column
equilibrated with distilled water and 0.1% (v/v) trifluoroacetic
acid (TFA) and tested for purity (FIG. 2A). Bound protein was
eluted with a gradient of acetonitrile in TFA (Van Patten et al.,
J. BIOL. CHEM., 274: 10268-76 (1999)). Additionally, the purity of
the final samples were assessed by SDS-polyacrylamide gel
electrophoresis (PAGE) with silver staining, and the protein
concentration was determined with a Bio-Rad protein assay.
Fractions with >95% purity by C4-HPLC and silverstaining were
used for the inhibition tests to determine the ID.sub.50 (Schreuder
et al., NAT, STRUCT. BIOL., 1: 48-54 (1994)).
[0141] The fractions from the Superdex-200 column that contained
anti-HIV activity were pooled and analyzed by SDS-PAGE under
reducing and non-reducing conditions and silver stain, which
revealed a single molecular species migrating at a 43 kDa (FIG.
2B). In-gel trypsin digestion was performed on the material
migrating at 43 kDa band to yield peptide fragments that were
subsequently eluted from the gel and identified as bovine ATIII by
reverse-phase HPLC nano-electrospray tandem mass spectrometry
(.mu.LC/MS/MS) on a Finnigan LCQ quadrupole ion trap mass
spectrometer (FIG. 2C). Bovine ATIII (53%) was detected with masses
of 14 peptides.
[0142] In contrast, serum containing untreated media and
supernatants from unstimulated CD8.sup.+ T-cell grown in serum
containing serum did not substantially inhibit HIV.sub.IIIB
replication, even when applied to the heparin Sepharose column.
Additionally, using untreated serum containing media the 43 kDa
form of ATIII was not detected following heparin Sepharose
chromatography and Superdex200 chromatography by either SDS-PAGE
silverstaining or C4-HPLC. These data show that activated CD8.sup.+
T-cells modify ATIII into a form that is capable of inhibiting HIV,
ATIII unprocessed normally has a molecular weight of 54-65 kDa,
whereas the purified form was found at 43 kDa by SDS-PAGE. This led
us to hypothesize that the heparin non-binding <50 kDa factor
might be necessary to activate ATIII.
Example 2
Antiviral Activity of ATIII
[0143] 1. Comparative Evaluation of the Effect of Purified Bovine
ATIII Forms on HIV, SIV, and SHIV Infectivity In Vitro
[0144] To test the effect of ATIII on lentivirus infectivity (X4
HIV, R5 HIV, SIV.sub.239 or SHIV.sub.KU-1), cell lines (H9, PM1,
SEM-174) were cultured in the presence or absence of the various
forms of ATIII for up to nine days (Cf., FIGS. 3 and 4). Every
three days (days 3, 6, and 9), 1 ml cell supernatant was removed
from test wells and replaced with an equal volume of R20 culture
medium containing either bovine ATIII or human ATIII. Control wells
were similarly sampled, but received media without the ATIII
supplement.
[0145] Both the test and the control wells were again sampled on
the ninth day of culture and the concentration of the viral core
protein p24 (gag) for the HIV (Alliance.RTM. HIV-1 p24 ELISA kit;
NEN.RTM. Life Science, Boston, Mass., USA) or p27 antigen (SIV core
antigen ELISA kit, Coulter, Miami, Fla.) was measured for HIV and
SIV or SHIV infected cells, respectively. Inhibition of viral
replication in the test samples was calculated as a percentage of
p24 immunoreactivity observed in control wells.
[0146] 2. In Vivo Evaluation of ATIII Antiretroviral Activity.
[0147] Human cell lines can be cultivated in hollow fibers in the
subcutaneous and intraperitoneal compartments of mice (Hollingshead
et al., LIFE SCi., 57: 131-41 (1995)). In vivo evaluation of ATIII
antiretroviral activity can be evaluated in the murine hollow fiber
model developed by Hollingshead and coworkers (ANTIVIRAL REs., 28:
256-79 (1995)).
[0148] H9 or PM1 cell-bearing polyvinylidene fluoride fibers
(500,000 Mw cutoff; 1 mm I.D.: Spectrum Medical Corp., Houston,
Tex., USA) are prepared by filling conditioned hollow fibers with
cell inoculum (uninfected cells, acutely HIV infected cells or
chronically HIV infected cells) (Hollingshead et al., LIFE SCI.,
57, 131-41 (1995)). These inoculated hollow fibers are surgically
implanted either subcutaneously or in the peritoneal cavity of SCID
mice (SCID/NCr; NCI Animal Production Facility, NCI-FCRDC,
Frederick, Md., USA). Hollow-fiber-bearing SCID mice are dosed
either acutely or chronically with increasing amounts of purified
ATIII preparation. The ATIII preparation (3-500 U/mouse/day) are
administered to the hollow-fiber-bearing SCID mice by subcutaneous
injection, intraperitoneal injection, intravenous or oral routes.
At select times, blood is sampled from control and test animals and
serum prepared. The amount of viral particles in test and control
serum is measured by p24 ELISA. ATIII-mediated antiviral action
yield a significant decrease in viral load as judged by at least a
15% decrease in serum p24 protein content in ATIII-treated animals
relative to the serum p24 content of the untreated control
animals.
Example 3
Anti-Viral Activity of Modified ATIII
1. Viruses Tested
[0149] The NIH Biodefense program tested anti-viral activity of
AB100 and AB200 (ATIII-heparin complex; i.e. modified ATIII) for
the following viruses: HSV-1, HSV-2, Measles VEE, Tacarible Virus,
SARS, Rift Valley Fever (MP-12), RSV A, Rhinovirus, PIV, FluA
(H1N1, H3N2, H5N1, H1N1, H3N2, H5N1), Flu B. New Guinea virus,
Adenovirus (65089, Chicago), WNV and Dengue (New Guinea C), Raymond
Chung at Gastrointestinal Unit at the Massachusetts General
Hospital tested HCV anti-viral activity. Anti-viral HIV activity
was measured in the laboratory of Bruce Walker at the AIDS Research
Center at the Massachusetts General Hospital.
2. Anti-Viral Activity
[0150] Anti-viral activities were found against HSV-1, HSV-2, HCV
and HIV using modified ATIII. Using the CPE assay with HFF cells
inhibition was found for the HSV-2 virus with a CC50 of 50 ug/ml.
The SI was above factor 3125 and the ACV EC50 was 0.3. Modified
ATIII had an EC50 of 3.57 pM for HSV-2. Here the EC90 was 5.36 pM,
the CC50>890 nM (>50 .mu.g/ml), a SI>250 with an ACV EC50
of 0.3. HCV anti-viral activity was measured in a replicon system
with an EC50 of 4.2 .mu.M (235 .mu.g/ml). For HIV an EC50 of 84 nM
was found where modified AT3 was given every 3 days and virus was
measured at day 9.
3. Summary of Anti-Viral Tests Used where Modified ATIII Showed
Anti-Viral Activity 3.1 HSV-1/HSV-2 test as described at Website
niaid-aacf.org/protocols/Herpes 3.1.1 Efficacy. In all the assays,
a minimum of six drug concentrations was used covering a range of
100 .mu.g/ml to 0.016 .mu.g/ml, in 5-fold increments. These data
allow to obtain good dose response curves. From these data, the
dose that inhibited viral replication by 50% (effective
concentration 50; EC.sub.50) was calculated using the computer
software program MacSynergy II by M. N. Prichard, K. R. Asaltine,
and C. Shipman, Jr., University of Michigan, Ann Arbor, Mich. 3.1.2
Toxicity. The same drug concentrations used to determine efficacy
were also used on uninfected cells in each assay to determine
toxicity of each experimental compound. The drug concentration that
is cytotoxic to cells as determined by their failure to take up a
vital strain, neutral red, (cytotoxic concentration 50; CC.sub.50)
was determined as described above.
[0151] Since the greatest need for new drugs to treat herpes virus
infections are for systemic diseases such as neonatal herpes, it is
likely that these drugs will need to be given parenterally. It is
very important therefore to determine the toxicity of new compounds
on dividing cells at a very early stage of testing. A cell
proliferation assay using HFF cells is a very sensitive assay for
detecting drug toxicity to dividing cells and the drug
concentration that inhibits cell growth by 50% (IC.sub.50) was
calculated as described above. In comparison with four human
diploid cell lines and vero cells, HFF cells are the most sensitive
and predictive of toxicity for bone marrow cells.
3.1.3 Assessment of Drug Activity. To determine if each compound
has sufficient antiviral activity that exceeds its level of
toxicity, a selectivity index (SI) was calculated according to
CC.sub.50/EC.sub.50. A compound that had an SI of 10 or greater is
considered to have anti-viral activity. 3.2 HCV test (i.e. replicon
system) as described in Chung, R. T., W. He, A. Saquib, A. M.
Contreras, R. J. Xavier, A. Chawla. T. C. Wang, and E. V. Schmidt,
2001. Hepatitis C virus replication is directly inhibited by
IFN-alpha in a full-length binary expression system. Proc Natl Acad
Sci USA 98:9847-9852.
[0152] For the HCV anti-viral test a replicon system was used. 0,
10, 50 and 250 .mu.g/ml of modified ATIII was used in the test.
3.3.1.1 HIV test as described in Geiben-Lynn, R., M. Kursar, N. V.
Brown, E. L. Kerr, A. D. Luster, and B. D. Walker. 2001.
Noncytolytic inhibition of X4 virus by bulk CD8(+) cells from human
immunodeficiency virus type 1 (HIV-1)-infected persons and
HIV-1-specific cytotoxic T lymphocytes is not mediated by
beta-chemokines. J Virol 75:8306-8316. 3.3.1 Assay for inhibition
of HIV-I.sub.IIIB replication. H9 cells (HLA A1, B6, Bw62, Cw3)
were acutely infected with HIV-I.sub.IIIB at a multiplicity of
infection of 10.sup.2 50% tissue culture infective dose per ml and
resuspended in R20. The cells were then plated in 2 ml R20 at
5.times.10.sup.5 cells/ml in a 24-well plate. Modified ATIII
concentrations were tested at 0, 10, 50, 250 .mu.g/ml. H9 cell
supernatant (1 ml) was removed every 3 days and replaced with
medium supplemented with modified ATIII. After 9 days, the
concentration of p24 was measured with an HIV-1 p24 enzyme-linked
immunosorbent assay (ELISA) kit (NEM Life Science, Boston, Mass.),
and the percentage inhibition was calculated against the medium
control.
Example 4
Anti-Viral Activity of Activated ATIII
1. Viruses Tested
[0153] We tested antiviral activity of activated ATIII (ATIII after
interaction with heparin, but not still bound, i.e., S-ATIII) for
the following viruses: HSV-1, HSV-2, Measles VEE, Tacarible Virus,
SARS, Rift Valley Fever (MP-12). RSV A, Rhinovirus, PIV, FluA
(H1N1, H3N2, H5N1, H1N1, H3N2, H5N1), Flu B, New Guinea virus.
Adenovirus (65089, Chicago), WNV and Dengue (New Guinea C).
2. Anti-Viral Activity
[0154] Anti-viral activities were found against HSV-1 and HSV-2
using activated ATIII. Using the CPE assay with HFF cells strongest
inhibition was found for the HSV-1 virus with an EC50 of <0.6 pM
(<0.016 .mu.g/ml), an EC90 of <0.6 pM. The CC50 was 50
.mu.g/ml, the SI was above factor 3125 and the ACV EC50 was 0.3.
Using the same assay activated ATIII had an EC50 of 3.57 pM for
HSV-2. Here the EC90 was 5.36 pM, the CC50>890 nM (>50
.mu.g/ml), a SI>250 with an ACV EC50 of 0.3.
3. Summary of Anti-Viral Tests Used where Activated ATIII Showed
Anti-Viral Activity 3.1 HSV-1/HSV-2 test as described at website
niaid-aacf.org/protocols/Herpes 3.1.1 Efficacy. In all the assays,
a minimum of six drug concentrations was used covering a range of
100 .mu.g/ml to 0.016 .mu.g/ml, in 5-fold increments. These data
allow to obtain good dose response curves. From these data, the
dose that inhibited viral replication by 50% (effective
concentration 50; EC.sub.50) was calculated using the computer
software program MacSynergy II by M. N. Prichard, K. R. Asaltine,
and C. Shipman, Jr., University of Michigan, Ann Arbor, Mich. 3.1.2
Toxicity. The same drug concentrations used to determine efficacy
were also used on uninfected cells in each assay to determine
toxicity of each experimental compound. The drug concentration that
is cytotoxic to cells as determined by their failure to take up a
vital strain, neutral red, (cytotoxic concentration 50; CC.sub.50)
was determined as described above.
[0155] Since the greatest need for new drugs to treat herpes virus
infections are for systemic diseases such as neonatal herpes, it is
likely that these drugs will need to be given parenterally. It is
very important therefore to determine the toxicity of new compounds
on dividing cells at a very early stage of testing. A cell
proliferation assay using HFF cells is a very sensitive assay for
detecting drug toxicity to dividing cells and the drug
concentration that inhibits cell growth by 50% (IC.sub.50) was
calculated as described above. In comparison with four human
diploid cell lines and vero cells, HFF cells are the most sensitive
and predictive of toxicity for bone marrow cells.
3.1.3 Assessment of Drug Activity. To determine if each compound
has sufficient antiviral activity that exceeds its level of
toxicity, a selectivity index (SI) was calculated according to
CC.sub.50/EC.sub.50. A compound that had an SI of 10 or greater is
considered to have anti-viral activity.
Example 5
Use of Serpin Drugs for Treatment of HIV/HCV Co-Transfections
[0156] Novel antivirals against HIV and HCV targeting host-cell
proteins are needed to prevent the occurrence of multi-resistant
viruses. The Serine protein inhibitors (Serpins) Secretory
Leucocyte Protease Inhibitor (SLPI), anti-trypsin and Antithrombin
III (ATIII) have potent antiviral activity against HIV in vitro.
Their in vivo potential can be seen in the facts: a) the missing
oral transmission most likely due to the anti-viral activity of
SLPI, the predominant HIV-inhibitor in saliva; b) the correlation
between disease progression and certain anti-trypsin mutations; c)
the observation that CD8.sup.+ T cell of HIV Long-term
Non-progressors (patients infected for more than 10 years but show
no signs of disease progression without anti-viral treatment)
produce a modified form of ATIII with high anti-viral activities.
ATIII is the first recombinant protein produced in goats and
approved for human use. Due to its improved availability, 60 h
half-life and low toxic profile ATIII offers new applications as an
anti-viral against HIV and HCV.
[0157] HIV inhibition was measured in cell lines and human
peripheral blood monocytes cells. HCV inhibition was measured using
a replicon system. Activation or inhibition of pathways and
host-cell target was measured by microarray with 84 key genes
testing for 14 different pathways.
[0158] ATIII blocked HIV viral replication in nM and HCV in .mu.M
concentrations in a dose dependent manner. Using 2.4, 12 and 24
U/ml ATIII we saw 8 genes in HIV infected PBMC up-regulated (PTGS2,
IL-8, IL-1.alpha., CCL20, BCL2A1, MMP7, Fas and HK2). At the
highest dose PTGS2 was up to 300 times, IL-8 up to 60 times and
IL-1.alpha. up to 60 times up-regulated whereas PECAM1 and IL-2
were down-regulated, 7 and 3 times respectively. In the HCV
replicon system seven genes were more than 10 times down-regulated.
Cancer genes Jun and Myc were up to 1000 times and 80 times
up-regulated, respectively, transcription factor CEBP was
up-regulated more than 600 times.
[0159] Whereas HIV is possibly blocked due to up-regulation of
proteins like the anti-inflammatory prostaglandins, HCV is
down-regulated through proteins necessary for viral replication.
ATIII blocks viral replication through an anti-viral mechanism of
action which targets host-cell proteins which might diminish the
ability of the viruses to gain resistance to the new treatment.
Example 6
Anti-HLA-DR Immunoliposomes Encapsulating Serine Protease
Inhibitors (Serpins) Targeting Virus-Infected Cells
[0160] Current small molecule anti-HIV drugs are targeting viral
structures in the replication cycle of HIV. Combinations of the
various small-molecule drugs are used to delay the emergence of
resistant viral variants, to increase potency and to broaden
coverage against variants that exist in the population.
Nevertheless, new antiretroviral drug regimens are needed,
especially for subjects who have failed two or three previous
regimens. Furthermore, to prevent further development of drug
resistant HIV a new class of drugs is needed targeting host-cell
proteins, which are necessary for the virus to replicate. These
targets are less mutable, possibly making it more difficult for the
virus to evolve resistance. In addition to the teachings herein, a
number of recent clinical observations point to the possibility of
the anti-inflammatory serine protease inhibitors (serpins) in
controlling viral infections in the mucosa and the peripheral
blood, possibly by blocking pathways necessary for HIV to
replicate.
[0161] In this example antithrombin III (ATIII) was investigated as
a prototypic member of this family of proteins as it displays up to
10.sup.6-fold higher in vitro anti-HIV activity than other serpins.
HIV replicates only slowly in whole blood, but more rapidly in
.alpha.1-antitrypsin deficient blood. Therefore, heparin activated
ATIII (hep-ATIII) encapsulated in liposomes was used to target
specifically the lymph nodes because the peripheral blood might
already be saturated with serpins. Conventional liposomes were
compared with sterically stabilized anti-HLA-DR immunoliposomes.
Monocyte-derived macrophages, which are also CD4.sup.+ and express
HLA-DR, are the most frequent hosts of HIV-1 in tissues of infected
individuals. Therefore, using HLA-DR antibodies engrafted into the
surface of sterically stabilized liposomes were used for improved
targeting.
[0162] For in vitro assays peripheral blood mononuclear cells
(PBMCs) from HIV-1-seronegative donors were obtained by
Ficoll-Hypaque density gradient centrifugation of heparinized
venous blood. After 3-day mitogen stimulation, PBMCs were
resuspended at a concentration of 1.times.10.sup.6 cells/ml in RPMI
1640 culture medium (Sigma, St Louis, Mo.) supplemented with 10%
fetal calf serum (Sigma), penicillin (50 U/ml), streptomycin (50
.mu.g/ml). L-glutamine (2 mM), HEPES buffer (10 mM), and 50 U/ml
interleukin-2 in 24-well tissue culture plates (Becton Dickinson,
San Jose, Calif.). Conventional and sterically stabilized
anti-HLA-DR liposomes encapsulating a heparin activated ATIII were
added in serial dilutions at day 0 and day 4. HIV-1 inoculum (1.000
50% tissue culture infective doses/10.sup.6 cells) was added to
PBMC for 2 h at 37.degree. C. and cells were washed extensively,
afterwards liposome preparations were added in serial dilutions.
Fifty percent of medium was replaced at day 4. Each condition was
tested in triplicate. Cell-free culture supernatants were harvested
and analyzed by an enzyme-linked immunosorbent assay (Du Pont,
Wilmington. DE) for HIV-1 p24 antigen production on day 7 of
culture. In addition, uninfected drug-treated cytotoxicity controls
were maintained at the highest concentration of each liposome
tested. No toxicity was observed at highest concentrations tested,
assessed by trypan blue dye exclusion method or Neutral Red
staining.
[0163] Two clinical HIV-1 isolates, HIV-1 89.6 and SF162, were
derived from PBMCs or cerebrospinal fluid of patients with AIDS,
respectively. 89.6 replicates to high titers in primary human
macrophages, primary human lymphocytes, CEMx174 and MT-2 cells. The
HIV-1 isolate is syneytium-forming and highly cytopathic. It
utilizes CCR5 and CXCR4 as co-receptors. Isolate SF162 is not
easily neutralized by antibodies, does not grow in T- or U937
cells. SF120 utilizes CCR5 as a coreceptor.
[0164] Before encapsulation ATIII (Talecris, Durham. NC) was
activated with heparin as previously described. The conventional
liposomes for the studies were made as large liposomes through a
mixture of phosphatidyl-choline and cholesterol, and were passed
through 5 .mu.m polycarbonate membrane filter to reach a final size
of 1-2 .mu.m. The sterically stabilized anti-HLA DR antibody (clone
2.06, IgG.sub.1) immunoliposomes were produced as described
earlier, with the modification that instead the Fab' fragment the
whole antibody was used. The immunoliposomes had a final size of
100 nm. Per subcutaneous administration on day 0 and day 1 a
delivery schedule of four equal depots of immunoliposomes (1.5 ml
in total) were delivered close to the inguinal or axillary lymph
nodes. The animals used were chronically infected for more than 450
days with SIV.sub.mac251 as previously described.
[0165] Two different liposome formulations encapsulating hep-ATIII
were examined and their effects on viral replication in vitro were
compared. Hep-ATIII encapsulated in sterically stabilized liposomes
increased anti-viral activity 20-50 times compared to conventional
liposomes (Table 1) with IC.sub.50 in a low nanomolar range. This
indicates that the use of immunoliposomes increased the efficiency
of delivery to the target cells.
[0166] The effect of the two different liposome formulations were
then compared in vivo experiments. Three animals treated with
conventional liposomes (0.8 mg/ml hep-ATIII encapsulated) showed no
effect on viral load. Animals treated with sterically stabilized
anti-HLA DR antibody immunoliposome encapsulating 0.05 mg/ml
hep-ATIII showed a 0.9-2.5 log reduction of viral load (FIG. 12).
This confirms to the in vitro findings that the target cell
specific delivery increased immunoliposomes efficiency.
[0167] As the microenvironment of lymphoid tissues is crucial for
effective immune response, it is important to decrease viral burden
and inhibit virus replication at the earliest possible time after
infection. Three-drug treatment therapy markedly diminished the
number of HIV-1 RNA copies found in the secondary lymphoid tissues
such as the tonsils. However, a few copies of HIV-1 RNA were still
detectable and could thus represent a focus of infection once the
therapy is stopped due to the frequent toxicity seen in patients
undergoing combined anti-retroviral therapy. As suboptimal
concentrations of drugs within infected cells can potentially lead
to the development of resistance, the delivery of high drug
concentrations into HIV reservoirs could also reduce the frequency
of resistance.
[0168] An immunoliposome formulation of the invention, can be used
periodically to deplete residual viral reservoirs or used routinely
in salvage patient populations, where resistant viruses prevail in
spite of a multiple-drug treatment therapy.
Rhesus Macaque Treatment:
[0169] For non-human primate studies, two Indian-origin macaques
were chronically infected with SIVmac251 for more than 450 days and
then treated each via s.c. route with 4 inoculations of a total of
1.5 ml of 0.05 mg/ml hep-ATIII encapsulated in anti-1HLA-DR
immunoliposomes daily for two days. S.C. administrations were done
in close proximity to the inguinal or axillary lymph nodes.
TABLE-US-00001 TABLE 1 IC.sub.50 of hep-ATIII encapsulated in
different liposome formulations IC.sub.50 Formulation Virus (nM)
Conventional 89.6 24 Liposome SF162 22 Anti-HLA-DR 89.6 0.4
Immunoliposome SF162 0.7
Immunoliposome Preparation:
[0170] Step 1 is the derivation of antibody with NGPE: 0.6 ml of
0.1 M of octyl glucoside (OG) was added to 6 ml of MES/NaC buffer.
3.30 mg of NOPE lipid (Sigma) was dissolved in 2 ml of chloroform
and added to a 50 ml round bottom flask. The chloroform was dried
in a rotary evaporator. The OG in MES buffer solution was added to
the dried film. Then 1.1 ml of 0.25 M EDC (Sigma) was added to the
solution. Afterwards, 1.1 ml of 0.1 M NHS was added and the
solution was incubated at room temperature for 10 minutes. The pH
was adjusted to 7.5. Fifty mg HLA-DR antibody (clone 2.06), which
was dialyzed against a sodium borate buffer, was added. The
solution was incubated for 12 hours at 4.degree. C. and afterwards
dialyzed against PBS buffer. The NGPE conjugated antibody was
concentrated in a vacuum concentrator. Step 2: 40 mg of DOPA and
164 mg of DOPE (both Sigma) were dissolved in 100 ml of chloroform
in a round bottom flask connected to a rotary evaporator. The
chloroform was evaporated by vacuum until a thin lipid film was
formed. The NGPE conjugated antibody and 1 ml hep-ATIII solution
(20 mg/ml) was added to the dried lipid film. This film was
hydrated overnight at 4.degree. C. during to form the liposomes.
The liposomes were sized to 100 nm by 20 times extrusion through a
5 micron polycarbonate membrane filter using a Lipex extruder. The
liposomes were then dialyzed against 10 L of PBS buffer for 36
h.
Example 7
Up-Regulation and Down-Regulation of Gene Expression
[0171] FIG. 13 shows Target genes of Affymetrix gene expression
profiling of genes activated after hep-ATIII and ATIII treatment.
Heat diagram of genes activated or down-regulated by heparin
activated, hep-ATIII, and ATIII is shown for three rhesus macaques.
Software analysis was performed using the Ingenuity 4.0 software
package. Only genes are shown which were significantly (p<0.01)
altered at day 7 compared to same animals before treatment. For the
analysis day 7 was used because here occurred the highest reduction
of viral load. For ATIII treatment macaques were treated daily for
4 days with 50 mg/ml daily by intra-venous administration, then
every other day for 14 days. PBMC were collected at day 14. For
hep-ATIII treatment macaques were treated by intra-venous injection
with 50 mg/ml once daily for 4 days. PBMC were isolated at day 7.
Total RNA was purified from these PBMC after shredding cells using
the QIAshredder homogenizer (Qiagen), which consists of a unique
biopolymer-shredding system in a microcentrifuge spin-column
format. Total RNA was purified using RNAeasy spin-columns (Qiagen)
according to the manufacturer's protocol. Integrity and
concentration or the RNA sample was tested using the Aglient
BioAnalyser and readied for the Affymetrix system setup consisting
of target preparation, target hybridization and fluidics station,
probe array washing and staining, and probe array scan. Probe was
analyzed by Ingenutity software.
Example 8
Use of Serpin Drugs for Treatment of H1N1 Viral Infection
Viral Inhibition Assay:
[0172] A. Visual or Visual-Confimation (Visual-CONF): Inhibition of
Viral Cytopathic Effect (CPE) This test, run in 96 well
flat-bottomed microplates, was used. In this CPE inhibition test,
four log 10 dilutions of ATIII (e.g. 1000, 100, 10, 1 .mu.g/ml)
were added to 3 cups containing the cell monolayer; within 5 min,
the virus is then added and the plate sealed, incubated at
37.degree. C. and CPE read microscopically when untreated infected
controls develop a 3 to 4+ CPE (approximately after 72 to 120 hr).
A known positive control drug is evaluated in parallel with test
drugs in each test. This drug was ribavirin. Follow-up testing
(Visual-CONF) was run in the same manner except 8 one-half log 10
dilutions of ATIII was used in 4 cups containing the cell monolayer
per dilution.
[0173] The data are expressed as 50% effective concentrations
(EC50).
B. Neutral Red Assay: Increase in Neutral Red (NR) Dye Uptake
[0174] This test was run to validate the CPE inhibition seen in the
initial test, and utilizes the same 96-well micro plates after the
CPE has been read. Neutral red was added to the medium; cells not
damaged by virus take up a greater amount of dye, which was read on
a computerized micro plate autoreader. The method as described by
McManus (Appl. Environment. Microbiol. 31:35-38, 1976) is used. An
EC50 was determined from this dye uptake.
C. Virus Yield Assay
[0175] ATIII effect on reduction of virus yield was tested by
assaying frozen and thawed eluates from each cup for virus titer by
serial dilution onto monolayers of susceptible cells. Development
of CPE in these cells is the indication of presence of infectious
virus. As in the initial tests, a known active drug was run in
parallel as a positive control. The 90% effective concentration
(EC90), which is that test drug concentration that inhibits virus
yield by 1 log 10, is determined from these data.
TABLE-US-00002 TABLE 2 Corp ID Assay Vehicle Virus Strain Cell Line
Unit Test Date Trial EC50 EC90 IC50 SI Anti- Neutral Company Flu A
(H1N1) California/07/2009 MDCK .mu.g/ml Jun. 23, 2009 1 4.4 >100
>23 thrombin Red Buffer Visual Company Flu A (H1N1)
California/07/2009 MDCK .mu.g/ml Jun. 23, 2009 1 1.8 >100 >5
Buffer Virus Company Flu A (H1N1) California/07/2009 MDCK .mu.g/ml
Jun. 30, 2009 2 1.82 >55 Yield Buffer Visual- Company Flu A
(H1N1) California/07/2009 MDCK .mu.g/ml Jun. 30, 2009 2 1.8 >100
>55 CONF Buffer indicates data missing or illegible when
filed
Table 2 shows data for MDCK cells infected with H1N1 virus and
treated with the serpin, antithrombin, of the present invention.
The dose that inhibited viral replication by 50% (90%) called
inhibitory concentration 50, EC50 (EC90) was calculated using the
MacSynergy II Software [98]. A screen for drug toxicity was
conducted in parallel. To determine if each compound has sufficient
antiviral activity that exceeds its level of toxicity, a
therapeutic index (SI) was calculated according to EC50/IC50. A
compound that had a SI of 10 or greater is considered to have high
anti-viral activity.
EQUIVALENTS
[0176] Although particular embodiments have been disclosed herein
in detail, this has been done by way of example for purposes of
illustration only, and is not intended to be limiting with respect
to the scope of the appended claims, which follow. In particular it
is contemplated by the inventors that various substitutions,
alterations, and modifications may be made to the invention without
departing from the spirit and scope of the invention as defined by
the claims. Other aspects, advantages, and modifications are within
the scope of the invention. For example, ATIII can be used, for
example, as an antiviral drug against other viruses (e.g., H1N1,
HTLV-1, HTLV-2, HSV-1, HSV-2, EBV, HBV, HCV, or CMV).
Sequence CWU 1
1
11433PRTHuman immunodeficiency virus 1His Arg Ser Pro Val Glu Asp
Val Cys Thr Ala Lys Pro Arg Asp Ile 1 5 10 15 Pro Val Asn Pro Met
Cys Ile Tyr Arg Ser Ser Glu Lys Lys Ala Thr 20 25 30 Glu Gly Gln
Gly Ser Glu Gln Lys Ile Pro Gly Ala Thr Asn Arg Arg 35 40 45 Val
Trp Glu Leu Ser Lys Ala Asn Ser His Phe Ala Thr Ala Phe Tyr 50 55
60 Gln His Leu Ala Asp Ser Lys Asn Asn Asn Asp Asn Ile Phe Leu Ser
65 70 75 80 Pro Leu Ser Ile Ser Thr Ala Phe Ala Met Thr Lys Leu Gly
Ala Cys 85 90 95 Asn Asn Thr Leu Thr Gln Leu Met Glu Val Phe Lys
Phe Asp Thr Ile 100 105 110 Ser Glu Lys Thr Ser Asp Gln Ile His Phe
Phe Phe Ala Lys Leu Asn 115 120 125 Cys Arg Leu Tyr Arg Lys Ala Asn
Lys Ser Ser Glu Leu Val Ser Ala 130 135 140 Asn Arg Leu Phe Gly Asp
Lys Ser Ile Thr Phe Asn Glu Thr Tyr Gln 145 150 155 160 Asp Ile Ser
Glu Val Val Tyr Gly Ala Lys Leu Gln Pro Leu Asp Phe 165 170 175 Lys
Gly Asn Ala Glu Gln Ser Arg Leu Thr Ile Asn Gln Trp Ile Ser 180 185
190 Asn Lys Thr Glu Gly Arg Ile Thr Asp Val Ile Pro Pro Gln Ala Ile
195 200 205 Asn Glu Phe Thr Val Leu Val Leu Val Asn Thr Ile Tyr Phe
Lys Gly 210 215 220 Leu Trp Lys Ser Lys Phe Ser Pro Glu Asn Thr Arg
Lys Glu Leu Phe 225 230 235 240 Tyr Lys Ala Asp Gly Glu Ser Cys Ser
Val Leu Met Met Tyr Gln Glu 245 250 255 Ser Lys Phe Arg Tyr Arg Arg
Val Ala Glu Ser Thr Gln Val Leu Glu 260 265 270 Leu Pro Phe Lys Gly
Asp Asp Ile Thr Met Val Leu Ile Leu Pro Lys 275 280 285 Leu Glu Lys
Thr Leu Ala Lys Val Glu Gln Glu Leu Thr Pro Asp Met 290 295 300 Leu
Gln Glu Trp Leu Asp Glu Leu Thr Glu Thr Leu Leu Val Val His 305 310
315 320 Met Pro Arg Phe Arg Ile Glu Asp Ser Phe Ser Val Lys Glu Gln
Leu 325 330 335 Gln Asp Met Gly Leu Glu Asp Leu Phe Ser Pro Glu Lys
Ser Arg Leu 340 345 350 Pro Gly Ile Val Ala Glu Gly Arg Ser Asp Leu
Tyr Val Ser Asp Ala 355 360 365 Phe His Lys Ala Phe Leu Glu Val Asn
Glu Glu Gly Ser Glu Ala Ala 370 375 380 Ala Ser Thr Val Ile Ser Ile
Ala Gly Arg Ser Leu Asn Ser Asp Arg 385 390 395 400 Val Thr Phe Lys
Ala Asn Arg Pro Phe Leu Val Leu Ile Arg Glu Val 405 410 415 Ala Leu
Asn Thr Ile Ile Phe Met Gly Arg Val Ala Asn Pro Cys Val 420 425 430
Asp
* * * * *