U.S. patent application number 12/790296 was filed with the patent office on 2011-01-27 for compositions, methods and uses for treating bacterial infections.
Invention is credited to Leland SHAPIRO.
Application Number | 20110021416 12/790296 |
Document ID | / |
Family ID | 34216145 |
Filed Date | 2011-01-27 |
United States Patent
Application |
20110021416 |
Kind Code |
A1 |
SHAPIRO; Leland |
January 27, 2011 |
COMPOSITIONS, METHODS AND USES FOR TREATING BACTERIAL
INFECTIONS
Abstract
A novel method of treating and preventing bacterial diseases is
provided. In particular, the present invention relates to
compositions and methods for inhibition of Gram negative, Gram
positive and acid fast bacilli in general and tuberculosis (TB),
mycobacterium avium complex (MAC), and anthrax in particular. Thus,
the invention relates to modulation of cellular activities,
including macrophage activity, and the like. More particularly, the
present invention relates to the inhibitory compounds comprising
naturally occurring and man-made inhibitors of serine protease.
Inventors: |
SHAPIRO; Leland; (Denver,
CO) |
Correspondence
Address: |
FAEGRE & BENSON LLP;PATENT DOCKETING - INTELLECTUAL PROPERTY
2200 WELLS FARGO CENTER, 90 SOUTH SEVENTH STREET
MINNEAPOLIS
MN
55402-3901
US
|
Family ID: |
34216145 |
Appl. No.: |
12/790296 |
Filed: |
May 28, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10926051 |
Aug 26, 2004 |
7850970 |
|
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12790296 |
|
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60497703 |
Aug 26, 2003 |
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Current U.S.
Class: |
514/2.8 ;
514/2.4; 514/21.6; 514/21.7 |
Current CPC
Class: |
A61P 43/00 20180101;
C07K 2319/50 20130101; C07K 14/811 20130101; C07K 14/473 20130101;
C07K 2319/21 20130101; C07K 16/00 20130101; C07K 2319/23 20130101;
A61K 38/57 20130101; A61P 31/04 20180101; A61K 45/06 20130101; A61K
38/57 20130101; A61K 31/519 20130101; C07K 14/8125 20130101; A61P
31/06 20180101; C07K 2319/30 20130101; C07K 2319/43 20130101; A61P
31/08 20180101; A61K 2300/00 20130101; A61K 2300/00 20130101; C07K
2319/00 20130101; A61K 31/519 20130101 |
Class at
Publication: |
514/2.8 ;
514/2.4; 514/21.6; 514/21.7 |
International
Class: |
A61K 38/08 20060101
A61K038/08; A61P 31/04 20060101 A61P031/04 |
Claims
1. A method for treating a bacterial infection in a subject
comprising: administering a therapeutically effective amount of a
composition comprising mammalian .alpha.1-antitrypsin or fragment
thereof, the .alpha.1-antitrypsin comprises naturally occurring
.alpha.1-antitrypsin, and treating the subject for the bacterial
infection.
2. The method of claim 1, wherein the naturally occurring
.alpha.1-antitrypsin is not a mutant form of
.alpha.1-antitrypsin.
3. The method of claim 1, wherein the composition comprising a
mammalian .alpha.1-antitrypsin is a pharmaceutical composition
comprising SEQ ID NO:61 or fragment thereof to the subject.
4. The method of claim 1, wherein the fragment comprises one or
more peptide selected from the group consisting of FVFLM (SEQUENCE
ID NO. 1), or analog of FVFLM, FVFAM (SEQUENCE ID NO. 2), FVALM
(SEQUENCE ID NO. 3), FVFLA (SEQUENCE ID NO. 4), FLVFI (SEQUENCE ID
NO. 5), FLMII (SEQUENCE ID NO. 6), FLFVL (SEQUENCE ID NO. 7), FLFVV
(SEQUENCE ID NO. 8), FLFLI (SEQUENCE ID NO. 9), FLFFI (SEQUENCE ID
NO. 10), FLMFI (SEQUENCE ID NO. 11), FMLLI (SEQUENCE ID NO. 12),
FIIMI (SEQUENCE ID NO. 13), FLFCI (SEQUENCE ID NO. 14), FLFAV
(SEQUENCE ID) NO. 15), FVYLI (SEQUENCE ID NO. 16), FAFLM (SEQUENCE
ID NO. 17), AVFLM (SEQUENCE ID NO. 18), and a combination
thereof.
5. The method of claim 1, wherein the mammalian
.alpha.1-antitrypsin comprises Aralast.TM. (Baxter), Zemaira.TM.
(Aventis Behring), Prolastin.TM. (Bayer), or a combination
thereof.
6. The method of claim 1, wherein the bacterial infection is caused
by one or more gram negative bacteria.
7. The method of claim 1, wherein the bacterial infection is caused
by one or more gram positive bacteria.
8. The method of claim 1, wherein administration comprises
continuous infusion to provide 0.01 to 5.0 mg/kg of
.alpha.1-antitrypsin to the subject.
9. The method of claim 1, wherein administration comprises
intermittent infusions comprising about 0.4 to 20 mg/kg of
.alpha.1-antitrypsin to the subject.
10. The method of claim 1, further comprising administering at
least one additional anti-bacterial agent to the subject.
11. A method for treating a bacterial infection in a subject
comprising: administering a therapeutically effective amount of a
composition comprising mammalian .alpha.1-antitrypsin or fragment
thereof, the .alpha.1-antitrypsin is naturally occurring
.alpha.1-antitrypsin and does not have furin endoprotease
inhibitory activity; and treating the subject for the bacterial
infection.
12. The method of claim 11, wherein the fragment comprises one or
more peptide selected from the group consisting of FVFLM (SEQUENCE
ID NO. 1), or analog of FVFLM, FVFAM (SEQUENCE ID NO. 2), FVALM
(SEQUENCE ID NO. 3), FVFLA (SEQUENCE ID NO. 4), FLVFI (SEQUENCE ID
NO. 5), FLMII (SEQUENCE ID NO. 6), FLFVL (SEQUENCE ID NO. 7), FLFVV
(SEQUENCE ID NO. 8), FLFLI (SEQUENCE ID NO. 9), FLFFI (SEQUENCE ID
NO. 10), FLMFI (SEQUENCE ID NO. 11), FMLLI (SEQUENCE ID NO. 12),
FIIMI (SEQUENCE ID NO. 13), FLFCI (SEQUENCE ID NO. 14), FLFAV
(SEQUENCE ID) NO. 15), FVYLI (SEQUENCE ID NO. 16), FAFLM (SEQUENCE
ID NO. 17), AVFLM (SEQUENCE ID NO. 18), and a combination
thereof.
13. The method of claim 11, wherein the mammalian
.alpha.1-antitrypsin comprises Aralast.TM. (Baxter), Zemaira.TM.
(Aventis Behring), Prolastin.TM. (Bayer), or a combination
thereof.
14. A method of treating a subject exposed or suspected of being
exposed to a pathogenic bacteria comprising, administering to the
subject a pharmaceutically effective amount of mammalian
.alpha.1-antitrypsin or fragment thereof, the .alpha.1-antitrypsin
or fragment thereof inhibits endogenous host protease cell-surface
processing of inactive large PA into active smaller PA molecule;
and treating the subject for the bacterial infection.
15. The method of claim 14, wherein the .alpha.1-antitrypsin or
fragment thereof comprises a protein or peptide having
Ala-Ile-Pro-Met corresponding to amino acids 355-358 of SEQ ID NO:
61.
16. A method for prolactically treating a subject for one or more
bacterial infection(s) in a subject comprising: administering a
therapeutically effective amount of a composition comprising
mammalian .alpha.1-antitrypsin or fragment thereof, the
.alpha.1-antitrypsin comprises naturally occurring
.alpha.1-antitrypsin, and prolactically treating the subject for
the bacterial infection.
17. A composition comprising, a peptide comprising AGAMFLEAIP
(SEQUENCE ID NO. 56); MSIPPEVKFN (SEQUENCE ID NO. 57); KPFVFLMIEQ
(SEQUENCE ID NO. 58); NTKSPLFMGK (SEQUENCE ID NO. 59); VVNPTQK
(SEQUENCE ID NO. 60), and a combination thereof.
18. The composition of claim 17 comprising, AGAMFLEAIP (SEQUENCE ID
NO. 56); MSIPPEVKFN (SEQUENCE ID NO. 57); KPFVFLMIEQ (SEQUENCE ID
NO. 58); and a combination thereof.
19. The composition of claim 17, wherein the composition comprises
fusion polypeptides.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of, and claims priority to,
application Ser. No. 10/926,051 filed Aug. 26, 2004, which claims
priority to provisional application Ser. No. 60/497,703 filed Aug.
26, 2003. Both applications are incorporated herein by reference in
their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions and methods
for inhibition of bacterial infections comprising Gram negative,
Gram positive, and acid fast bacilli in general and mycobacterium
tuberculosis (TB), mycobacterium avium complex (MAC), and anthrax
in particular, as well as to therapeutic treatment of diseases or
disorders that involve infection of macrophages. Thus, the
invention relates to modulation of cellular activities, including
macrophage activity, inhibition of toxin, and the like. More
particularly, the present invention also relates to inhibitory
compounds comprising naturally occurring and man-made serine
protease inhibitors and antagonists.
BACKGROUND OF THE INVENTION
[0003] Serine Proteases
[0004] Serine proteases serve an important role in human physiology
by mediating the activation of vital functions. In addition to
their normal physiological function, serine proteases have been
implicated in a number of pathological conditions in humans. Serine
proteases are characterized by a catalytic triad consisting of
aspartic acid, histidine and serine at the active site.
[0005] The naturally occurring serine protease inhibitors are
usually, but not always, polypeptides and proteins which have been
classified into families primarily on the basis of the disulfide
bonding pattern and the sequence homology of the reactive site.
Serine protease inhibitors, including the group known as serpins,
have been found in microbes, in the tissues and fluids of plants,
animals, insects and other organisms. Protease inhibitor activities
were first discovered in human plasma by Fermi and Pemossi in 1894.
At least nine separate, well-characterized proteins are now
identified, which share the ability to inhibit the activity of
various proteases. Several of the inhibitors have been grouped
together, namely .alpha.1-antitrypsin-proteinase inhibitor,
antithrombin III, antichymotrypsin, C1-inhibitor, and
.alpha.2-antiplasmin, which are directed against various serine
proteases, i.e., leukocyte elastase, thrombin, cathepsin G,
chymotrypsin, plasminogen activators, and plasmin. These inhibitors
are members of the .alpha.1-antitrypsin-proteinase inhibitor class.
The protein .alpha.2-macroglobulin inhibits members of all four
catalytic classes: serine, cysteine, aspartic, and
metalloproteases. However, other types of protease inhibitors are
class specific. For example, the .alpha.1-antitrypsin-proteinase
inhibitor (also known as (.alpha.1-antitrypsin or AAT) and
inter-.alpha.-trypsin inhibitor inhibit only serine proteases,
.alpha.1-cysteine protease inhibitor inhibits cysteine proteases,
and .alpha.1-anticollagenase inhibits collagenolytic enzymes of the
metalloenzyme class.
[0006] Human neutrophil elastase (NE) is a proteolytic enzyme
secreted by polymorphonuclear leukocytes in response to a variety
of inflammatory stimuli. The degradative capacity of NE, under
normal circumstances, is modulated by relatively high plasma
concentrations of .alpha.1-antitrypsin. However, stimulated
neutrophils produce a burst of active oxygen metabolites, some of
which (hypochlorous acid for example) are capable of oxidizing a
critical methionine residue in .alpha.1-antitrypsin. Oxidized
.alpha.1-antitrypsin has been shown to have a limited potency as a
NE inhibitor and it has been proposed that alteration of this
protease/antiprotease balance permits NE to perform its degradative
functions in localized and controlled environments.
[0007] .alpha.1-antitrypsin is a glycoprotein of MW 51,000 with 417
amino acids and 3 oligosaccharide side chains. Human
.alpha.1-antitrypsin was named anti-trypsin because of its
initially discovered ability to inactivate pancreatic trypsin.
Human .alpha.1-antitrypsin is a single polypeptide chain with no
internal disulfide bonds and only a single cysteine residue
normally intermolecularly disulfide-linked to either cysteine or
glutathione. The reactive site of .alpha.1-antitrypsin contains a
methionine residue, which is labile to oxidation upon exposure to
tobacco smoke or other oxidizing pollutants. Such oxidation reduces
the biological activity of .alpha.1-antitrypsin; therefore
substitution of another amino acid at that position, i.e. alanine,
valine, glycine, phenylalanine, arginine or lysine, produces a form
of .alpha.1-antitrypsin which is more stable. .alpha.1-antitrypsin
can be represented by the following formula:
TABLE-US-00001 MPSSVSWGIL LAGLCCLVPV SLAEDPQGDA AQKTDTSHHD
QDHPTFNKIT PNLAEFAFSL YRQLAHQSNS TNIFFSPVSI ATAFANLSLG TKADTHDEIL
100 EGLNFNLTEI PEAQIHEGFQ ELLRTLNQPD SQLQLTTGNG LFLSEGLKLV
DKFLEDVKKL YHSEAFTVNF GDHEEAKKQI NDYVEKGTQG KIVDLVKELD 200
RDTVFALVNY IFFKGKWERP FEVKDTEDED FHVDQVTTVK VPMMKRLGMF NIQHCKKLSS
WVLLMKYLGN ATAIFFLPDE GKLQHLENEL THDIITKFLE 300 NEDRRSASLH
LPKLSITGTY DLKSVLGQLG ITKVFSNGAD LSGVTEEAPL KLSKAVHKAV LTIDEKGTEA
AGAMFLEAIP MSIPPEVKFN KPFVFLMIEQ 400 NTKSPLFMGK VVNPTQK 417
[0008] Ciliberto, et al. in Cell 1985, 41, 531-540. The critical
amino acid sequence near the carboxyterminal end of
.alpha.1-antitrypsin is shown in bold and underlined and is
pertinent to this invention (details of the sequence can be found
for example in U.S. Pat. No. 5,470,970 as incorporated by
reference).
[0009] The normal plasma concentration of ATT ranges from 1.3 to
3.5 mg/ml although it can behave as an acute phase reactant and
increases 3-4-fold during host response to inflammation and/or
tissue injury such as with pregnancy, acute infection, and tumors.
It easily diffuses into tissue spaces and forms a 1:1 complex with
a target protease, principally neutrophil elastase. Other enzymes
such as trypsin, chymotrypsin, cathepsin G, plasmin, thrombin,
tissue kallikrein, and factor Xa can also serve as substrates. The
enzyme/inhibitor complex is then removed from circulation by
binding to serpin-enzyme complex (SEC) receptor and catabolized by
the liver and spleen. Humans with circulating levels of
.alpha.1-antitrypsin less than 15% of normal are susceptible to the
development of lung disease, e.g., familial emphysema, at an early
age. Familial emphysema is associated with low ratios of
.alpha.1-antitrypsin to serine proteases, particularly elastase.
Therefore, it appears that this inhibitor represents an important
part of the defense mechanism against attack by serine
proteases.
[0010] .alpha.1-antitrypsin is one of few naturally occurring
mammalian serine protease inhibitors currently approved for the
clinical therapy of protease imbalance. Therapeutic
.alpha.1-antitrypsin has been commercially available since the mid
80s and is prepared by various purification methods (see for
example Bollen et al., U.S. Pat. No. 4,629,567; Thompson et al.,
U.S. Pat. Nos. 4,760,130; 5,616,693; WO 98/56821). Prolastin is a
trademark for a purified variant of .alpha.1-antitrypsin and is
currently sold by Bayer Company (U.S. Pat. No. 5,610,285 Lebing et
al., Mar. 11, 1997). Recombinant unmodified and mutant variants of
.alpha.1-antitrypsin produced by genetic engineering methods are
also known (U.S. Pat. No. 4,711,848); methods of use are also
known, e.g., (.alpha.1-antitrypsin gene therapy/delivery (U.S. Pat.
No. 5,399,346 to French Anderson et al.).
[0011] The two known cellular mechanisms of action of serine
proteases are by direct degradative effects and by activation of
G-protein-coupled proteinase-activated receptors (PARs). The PAR is
activated by the binding of the protease followed by hydrolysis of
specific peptide bonds, with the result that the new N-terminal
sequences stimulate the receptor. The consequences of PAR
activation depend on the PAR type that is stimulated and on the
cell or tissue affected and may include activation of phospholipase
C.beta., activation of protein kinase C and inhibition of adenylate
kinase (Dery, O. and Bunnett, N. W. Biochem Soc Trans 1999, 27,
246-254; Altieri, D. C. J. Leukoc Biol 1995, 58, 120-127; Dery, O.
et al. Am J. Physiol 1998, 274, C1429-C1452).
[0012] TB and MAC
[0013] Mycobacterium is a genus of bacteria which are aerobic,
mostly slow growing, slightly curved or straight rods, sometimes
branching and filamentous, and distinguished by acid-fast staining.
Typically, mycobacteria are gram-positive obligate aerobes. The
genus mycobacterium includes the highly pathogenic organisms that
cause tuberculosis (M. tuberculosis and sometimes M. bovis) and
leprosy (M. leprae). There are, however, many other species of
mycobacterium such as M. avium-intracellulare, M. chelonei (also
known as borstelense and abscessus), M. africanum, M. marinium
(also known as balnei and platypoecilus), M. buruli (also known as
ulcerans), M. fortuitum (also known as giae, minetti, and ranae),
M. haemophilum, M. intracellulare, M. kansasii (also known as
luciflavum), M. littorale (also known as xenopi), M. malmoense, M.
marianum (also known as scrofulaceum and paraffinicum), M. simiae,
M. szulgai, and M. ulcerans.
[0014] Mycobacteria which are pathogenic for animals but not
believed to be pathogenic for humans include the following: M.
avium-intracellulare (also known as brunense), M. flavascens, M.
lepraemurium, M. microti, and M. paratuberculosis (which is the
causative agent for Johne's Disease, and perhaps Crohn's disease).
The following species of the genus mycobacterium are believed to be
non-pathogenic: M. gordonae (also known as aquae), M. gastri, M.
phlei (also known as moelleri and as timothy bacillus), M.
nonchromogenicum, M. smegmatis, M. terrae, M. triviale, and M.
vaccae.
[0015] Additionally, certain mycobacteria other than M.
tuberculosis and M. bovis are alternatively known as
non-tuberculosis mycobacteria. They are divided into four groups,
also known as Runyon groups, based on pigmentation and growth rate.
Each group includes several species. Group I refers to slow-growing
photochromogens; Group II refers to slow-growing scotochromogens;
Group III refers to slow-growing nonphotochromogens; and Group IV
refers to rapidly-growing mycobacteria. The non-tuberculosis
mycobacteria are also called atypical or anonymous
mycobacteria.
[0016] Tuberculosis is an acute or chronic infectious disease
caused by infection with M. tuberculosis. Tuberculosis is a major
disease in developing countries, as well as an increasing problem
in developed areas of the world, with approximately 8 million new
cases and 3 million deaths each year (See Styblo et al., Bull. Int.
Union Tuberc. 56:118-125 (1981). Although the infection may be
asymptomatic for a considerable period of time, the disease is most
commonly manifested as an acute inflammation of the lungs,
resulting in fever and a nonproductive cough. If left untreated,
serious complications and death typically result.
[0017] Although it is known that tuberculosis can generally be
controlled using extended antibiotic therapy, such treatment is not
sufficient to prevent the spread of the disease. Infected
individuals may be asymptomatic, but contagious, for some time. In
addition, although compliance with the specific treatment regimen
is critical, patient behavior is often difficult to monitor.
Treatment regimens often require six to twelve months of
uninterrupted therapy. As a result, some patients do not complete
the course of treatment, thus leading to ineffective treatment and
development of antibiotic resistance. Effective vaccination and
accurate, early diagnosis of the disease are needed in order to
inhibit the spread of tuberculosis. Vaccination with live bacteria
remains the most efficient method for inducing protective immunity.
The most common Mycobacterium employed in the live vaccine is
Bacillus Calmette-Guerin (BCG), an avirulent strain of
Mycobacterium bovis. Some countries, such as the United States,
however, do not vaccinate the general public because of concerns
regarding the safety and efficacy of BCG.
[0018] M. tuberculosis is an intracellular pathogen that infects
macrophages and is able to survive within the harsh environment of
the phagolysosome in macrophages. Most inhaled bacilli are
destroyed by activated alveolar macrophages. However, the surviving
bacilli multiply in macrophages and are released upon cell death,
which signals the infiltration of lymphocytes, monocytes and
macrophages to the site. Antigenic stimulation of T cells requires
presentation by MHC molecules. Lysis of the bacilli-laden
macrophages is mediated by the delayed-type hypersensitivity (DTH)
cell-mediated immune response and results in the development of a
solid caseous tubercle surrounding the area of infected cells.
Tuberculosis bacilli possess many potential T-cell antigens and
several have now been identified [Andersen 1994, Dan. Med. Bull.
41, 205]. Some of these antigens are secreted by the bacteria.
Continued DTH liquefies the tubercle, thereby releasing entrapped
tuberculosis bacilli. The large dose of extracellular tuberculosis
bacilli triggers further DTH, causing damage to the bronchi and
dissemination by lymphatic, hematogenous and bronchial routes, and
eventually allowing infectious bacilli to be spread by
respiration.
[0019] Cell-mediated immunity to tuberculosis involves several
types of immune effector cells. Activation of macrophages by
cytokines, such as interferon-.gamma., represents an effective
means of minimizing macrophage-based intracellular mycobacterial
multiplication. However, this does not lead to complete eradication
of the bacilli. Acquisition of protection against tuberculosis
additionally requires T lymphocytes. Among these, T cells of both
the CD8+ and CD4+ lineage appear to be particularly important [Orme
et al, 1993, J. Infect. Dis. 167, 1481]. These T-cells secrete
interferon-.gamma. in response to mycobacteria, indicative of a
T.sub.h 1 immune response, and possess cytotoxic activity to
mycobacteria-pulsed target cells. In recent studies using.beta.-2
microglobulin- and CD8-deficient mice, cytotoxic T lymphocyte (CTL)
responses have been shown to be critical in providing protection
against M. tuberculosis [Flynn et al, 1992, Proc. Natl. Acad. Sci.
USA 89, 12013; Flynn et al, 1993, J. Exp. Med. 178, 2249; Cooper et
al, 1993, J. Exp. Med. 178, 2243]. In contrast, B lymphocytes do
not appear to be involved, and passive transfer of
anti-mycobacterial antibodies does not provide any protective
immunity. Thus, an effective vaccine regimen against tuberculosis
must trigger cell-mediated immune responses.
[0020] Although commonly thought of only as a pulmonary infection,
TB is well known to afflict many parts of the body. In addition to
pulmonary TB, examples of other foci of tubercular infection
include miliary TB (generalized hematogenous or lymphohematogenous
TB), central nervous system TB, pleural TB, TB pericarditis,
genitourinary TB, TB of the gastrointestinal tract, TB peritonitis,
TB of the adrenals, TB of the liver, TB of the bones and joints
(for example, TB spondylitis or Pott's Disease), TB lymphadenitis,
and TB of the mouth, middle ear, larynx, and bronchial tree.
[0021] Conventional therapy for TB includes treatment with regimens
containing pyrazinamide, isoniazid, ethambutol, streptomycin,
rifampin, rifabutin, clarithromycin, ciprofloxacin, clofazamine,
azithromycin, ethionamide, amikacin and resorcinomycin A. To treat
latent (inactive) TB infection, isoniazid may be used alone.
However, the usual initial treatment for pulmonary tuberculosis
includes isoniazid in combination with at least one other drug,
such as ethambutol, streptomycin, rifampin or ethionamide.
Retreatment of pulmonary tuberculosis typically involves drug
combinations including rifampin and other drugs as noted above.
Development of resistance of the causative agent to anti-TB drugs,
especially isoniazid, is well known. Extrapulmonary tuberculosis is
also usually treated with a combination including rifampin and at
least one of the other three drugs mentioned.
[0022] Mycobacterium avium Complex (MAC)
[0023] M. avium and M. intracellulare are members of the
Mycobacterium avium complex (MAC). M. paratuberculosis is a
subspecies of M. avium and is also generally included in the MAC.
These species have become increasingly important in recent years
because of the high prevalence of disseminated MAC infection in
AIDS patients. The Mycobacterium avium complex is comprised of 28
serovars which are distinguishable on the basis of their
biochemical and seroagglutination characteristics (see review by
Inderlied, et al. 1993. Clin. Microbial. Rev. 6, 266-310).
Depending on the method of classification, 10-12 of the 28 serovars
are classified as belonging to the species Mycobacterium avium, and
10-12 belong to the species Mycobacterium intracellulare. Six of
the MAC serovars have not yet been definitively classified. MAC
infections currently account for approximately 50% of the
pathogenic isolates identified by mycobacteriology labs and are
most common among AIDS and other immuno-compromised patients. Early
diagnosis and treatment of MAC infections can improve and prolong
the lives of infected individuals.
[0024] Anthrax and Anthrax Toxin
[0025] Anthrax toxin, produced by the gram positive rod-shaped
aerobic, spore-forming bacterium Bacillus anthracis, is the toxic
virulence factor secreted by this organism. B. anthraxis is often
considered for use as a biological weapon due to the potency of the
secreted exotoxin, and to the capacity of the bacterium to form
dormant spores which resist harsh environmental conditions.
Sporulation enables ready transport and distribution of large
quantities of toxin-producing bacteria. The toxin is actually a
composite consisting of 3 separate secreted proteins from the
bacterium. The 3 proteins are protective antigen (PA), lethal
factor (LF), and edema factor (EF). While LF and EF directly damage
cells and cause disease, the PA is the focus of this disclosure. PA
is crucial to the virulence of anthrax toxin, since the PA molecule
is designed to import both LF and EF inside the membranes of cells.
In the absence of PA-induced intracellular transport, anthrax toxin
is unable to effect tissue destruction, since LF and EF only
function from within the cell. The importance of PA in the function
of anthrax toxin is underscored by the effective use of PA as the
immunogen in anthrax vaccine. By generating an immune response
against PA, the vaccine confers protection against full (3
component) anthrax toxin.
[0026] A closer examination of the interaction between PA and the
host cells attacked by anthrax toxin is instructive. PA is first
secreted as an 83 kDa monomeric polypeptide by B. anthracis in a
large and functionally inactive form. This inactive PA binds to a
mammalian receptor on the surface of host cells. The PA receptor
has recently been isolated and sequenced, and found to possess von
Willebrand Factor-like regions. After docking on the surface of
host cells, PA interacts with a protease present on the cell
surface. The protease processes the large and inactive PA molecule
into a smaller and active 63 kDa fragment. The C-terminal 63 kDa
fragment (PA63) remains bound to the cell and the N-terminal 20 kDa
(PA20) dissociates from PA63. The identity of the protease has been
the focus of scant research effort, and it is poorly characterized.
However, prior studies have shown that the protease has
characteristics that suggest it is a host-derived serine protease.
A possible serine protease candidate noted in the literature is
related to furine (itself a serine protease), but other serine
proteases, such as elastase, proteinase-3, clostripain, or trypsin
are possible alternatives (Molloy, S. S. et al. J Biol Chem 267,
16396-16402 (1992)). This proteolytic cleavage and subsequent
dissociation of PA20 confer two new properties on PA63: (1) the
ability to oligomerize into a ring-shaped heptameric
SDS-dissociable structure termed prepore and (2) the ability to
bind EF and LF. Oligomers containing PA63-EF, PA63-LF, or a
combination of PA63-EF and PA63-LF are endocytosed and trafficked
to an acidic compartment, where the PA63 prepore inserts into the
membrane and forms a pore. During or after pore formation, EF and
LF are translocated across the endosomal membrane into the
cytoplasm. EF is a calmodulin-dependent adenylate cyclase which may
protect the bacteria from destruction by phagocytes. LF is a
metalloprotease that can kill macrophages or, at lower
concentrations, induce macrophages to overproduce cytokines,
possibly resulting in death of the host. These heptamers function
as the transport vehicle to deliver LF and EF inside of the cell.
Once inside the cell, LF and EF initiate abnormalities in cell
function.
[0027] Because of some of the difficulties and inadequacies of
conventional therapy for tuburculosis, other mycobacterial
infections, and anthrax, new therapeutic modalities are
desirable.
[0028] The inventor discloses a novel method of use for serine
protease inhibitors as therapeutic agents to treat infections
caused by tuberculosis (TB) and mycobacterium avium complex (MAC).
These are intracellular human pathogens that establish infection
and prolonged latency by infecting and surviving within human
macrophages. Therefore, blocking the internalization of TB or MAC
within macrophages is a novel approach to therapy vs these
infectious agents. In an infectivity assay, the inventors have
shown that .alpha.1-antitrypsin significantly inhibited both TB and
MAC infection of human monocyte-derived-macrophages (MDM).
[0029] A novel approach to nullify the action of anthrax toxin is
to block access of the toxin to the interior of the cell by
interfering with the action of the host-derived serine protease
that resides on the cell surface.
[0030] This invention thus addresses a long-felt need for safe and
effective methods of treatment of tuberculosis, other mycobacterial
infections, other Gram negative and Gram positive bacterial
infections, and anthrax.
SUMMARY OF THE INVENTION
[0031] The present invention provides methods for treating
bacterial infections in a mammal comprising administering to a
subject in need thereof of a therapeutically effective amount of a
composition comprising a substance exhibiting mammalian
.alpha.1-antitrypsin or inhibitor of serine protease activity or a
functional derivative thereof; and a pharmaceutically acceptible
excipient.
[0032] In one embodiment, the bacterial infections that may be
treated or ameliorated using the compositions and methods of the
invention are those infections caused by Gram negative bacterial
organisms comprising N. gonorrhoeae, N. meningitidis, M.
catarrhalis, H. influenzae, E. coli, all Klebsiela spp., all
Enterobacter spp., all Serratia spp., all Salmonella spp., all
Shigella spp., Proteus mirabilis, Proteus vulgaris, all Providencia
spp., all Morganella spp., all Citrobacter spp., all Aeromonas
spp., all Acinetobacter spp., Pseudomonas aeruginosa, all
Pasteurella spp., Pseudomonas cepacia, Stenotrophomonas
maltophilia, Y. enterocolitica and other Yersinoiiosis, all
Legionella spp., P. multocida, H. ducreyeii, all Chlamyidia spp.,
Mycoplasma pneumoniae, Mycoplasma hominis, Bacteroides fragilis, P.
melaminogenica, all Moraxella spp., all Bortedella spp., or any
combination thereof.
[0033] In another embodiment, the bacterial infections that may be
treated or ameliorated using the compositions and methods of the
invention are those infections caused by Gram positive bacterial
organisms comprising C. tetani, C. botulinum, C. difficile, Group
A, B C, and G Streptococcus, Streptococcus pneumoniae,
Streptococcus milleri group, Viridans streptococcus, all Listeria
spp., all Staphylococcus spp., S. aureus (MSSA), S. aureus (MRSA),
S. epidermidis, Enterococcus faecalis, Enterococcus faecium, all
Clostridium spp. including C. diptheriea, C. jeikium, all
Rhodococcus spp., all Leukonostoc spp. or any combination
thereof.
[0034] In yet another embodiment, the bacterial infections that may
be treated or ameliorated using the compositions and methods of the
invention are those infections caused by acid fast bacilli
comprising Mycobacterium tuberculosis, and atypical Mycobacteria
(M. Avium, M. Intracellulare, M. Kansasii, M. Chelonei, M.
fortuitum, M. scrofulaceum, M. ulceranis, M. leprae, M. xenopi, M.
bovis, M. gordonae, M. haemophilum, M. marinum, M. genavense, M.
avium and intracellulari, and M. simiae), or any combination
thereof.
[0035] The present invention provides methods for treating
mycobacterial infections in a mammal comprising administering to a
subject in need thereof a therapeutically effective amount of a
composition comprising a substance exhibiting mammalian
.alpha.1-antitrypsin or inhibitor of serine protease activity or a
functional derivative thereof; and a pharmaceutically acceptible
excipient.
[0036] In one embodiment, the mycobacterium inhibited from
infecting macrophages comprises a mycobacterium from the genus
mycobacterium that includes M. tuberculosis M. bovis, M. leprae, M.
avium-intracellulare, M. chelonei (also known as borstelense and
abscessus), M. africanum, M. marinium (also known as balnei and
platypoecilus), M. buruli (also known as ulcerans), M. fortuitum
(also known as giae, minetti, and ranae), M. haemophilum, M.
intracellulare, M. kansasii (also known as luciflavum), M.
littorale (also known as xenopi), M. malmoense, M. marianum (also
known as scrofulaceum and paraffinicum), M. simiae, M. szulgai, M.
ulcerans, M. avium (also known as brunense), M. flavascens, M.
lepraemurium, M. microti, and M. paratuberculosis (which is the
causative agent for Johne's Disease), M gordonae (also known as
aquae), M. gastri, M phlei (also known as moelleri and as timothy
bacillus), M. nonchromogenicum, M. smegmatis, M. terse, M.
triviale, and M. vaccae, or any combination thereof.
[0037] In another embodiment, the mycobacterium inhibited from
infecting macrophages comprises a mycobacterium from the genus
mycobacterium that includes non-tuberculosis mycobacteria that are
divided into four groups comprising Runyon groups, selected from
the group consisting of Group I (slow-growing photochromogens),
Group II (slow-growing scotochromogens), Group III (slow-growing
nonphotochromogens), and Group IV (rapidly-growing mycobacteria),
or any combination thereof.
[0038] Therefore, in one aspect, the present invention provides
methods of treating mycobacterial diseases dependent on the
infection of macrophages.
[0039] Also provided is a method of inhibiting mycobacterial
infection of macrophages, which comprises administering to a mammal
susceptible to mycobacterial infection of macrophages an effective
amount of a substance exhibiting mammalian .alpha.1-antitrypsin or
inhibitor of serine protease activity. Without limiting to
.alpha.1-antitrypsin, the substance may be a compound that inhibits
proteinase-3, cathepsin G, elastase, or any other serine
protease.
[0040] In a preferred embodiment the agent that inhibits
mycobacterial infection of human monocyte-derived-macrophages
comprises .alpha.1-antitrypsin. In addition, peptides of interest
are homologous and analogous peptides. While homologues are natural
peptides with sequence homology, analogues will be peptidyl
derivatives, e.g., aldehyde or ketone derivatives of such peptides.
Typical examples of analogues are TLCK or TPCK. Without limiting to
.alpha.1-antitrypsin and peptide derivatives of
.alpha.1-antitrypsin, compounds like oxadiazole, thiadiazole,
CE-2072, UT-77, and triazole peptoids are preferred.
[0041] The agent that inhibits mycobacterial infection of human
monocyte-derived-macrophages can also be an inhibitor of serine
protease activity, an inhibitor of elastase, or an inhibitor of
proteinase-3. The inhibitor of serine protease activity can
include, but is not limited to, small organic molecules including
naturally-occurring, synthetic, and biosynthetic molecules, small
inorganic molecules including naturally-occurring and synthetic
molecules, natural products including those produced by plants and
fungi, peptides, variants of .alpha.1-antitrypsin, chemically
modified peptides, and proteins. An inhibitor of serine protease
activity has the capability of inhibiting the proteolytic activity
of trypsin, elastase, kallikrein, and/or other serine
proteases.
[0042] Also contemplated within the scope of the present invention
is a method of preventing a deficiency of functional endogenous
.alpha.1-antitrypsin levels in a patient susceptible to a
mycobacterial infection of macrophages that is mediated by
endogenous host serine protease or serine protease-like activity,
by treating with a pharmaceutical composition in a pharmaceutically
acceptable carrier comprising effective amounts of a substance
exhibiting mammalian .alpha.1-antitrypsin or inhibitor of serine
protease activity. The pharmaceutical composition can be a peptide
or a small molecule, which exhibits .alpha.1-antitrypsin or
inhibitor of serine protease activity.
[0043] In yet another aspect, the present invention provides a
method for preventing a symptom of anthrax in a subject thought to
be at risk for exposure to Bacillus anthracis comprising
administering to the subject a pharmaceutically effective amount of
a substance exhibiting mammalian "1-antitrypsin or inhibitor of
serine protease activity, wherein said mammalian "1-antitrypsin or
inhibitor of serine protease activity substance inhibits the
endogenous host protease cell-surface processing of inactive large
PA into the active smaller PA molecule, and wherein if the subject
is exposed to Bacillus anthracis, a symptom of said exposure is
prevented.
[0044] In another aspect, the present invention provides a method
for preventing a symptom of anthrax in a subject suspected of
having been exposured to Bacillus anthracis comprising
administering to the subject a pharmaceutically effective amount of
a substance exhibiting mammalian .alpha.1-antitrypsin or inhibitor
of serine protease activity, wherein said mammalian
.alpha.1-antitrypsin or inhibitor of serine protease activity
substance inhibits the endogenous host protease cell-surface
processing of inactive large PA into the active smaller PA
molecule, and wherein if the subject is exposed to Bacillus
anthracis, a symptom of said exposure is prevented.
[0045] In another aspect, the present invention provides a method
for ameliorating a symptom of anthrax in a subject in need of said
amelioration comprising administering to the subject a
pharmaceutically effective amount of a substance exhibiting
mammalian .alpha.1-antitrypsin or inhibitor of serine protease
activity, wherein said mammalian .alpha.1-antitrypsin or inhibitor
of serine protease activity substance inhibits the endogenous host
protease cell-surface processing of inactive large PA into the
active smaller PA molecule.
[0046] In the above-recited methods, the symptom of anthrax that is
inhibited or prevented is selected from the group consisting of
cutaenous ulceration, edema, and escar formation, or any
combination thereof.
[0047] In one embodiment, the methods of the present invention are
used to prevent or ameliorate a symptom of cutaneous,
gastrointestinal, and/or inhalation anthrax. In one embodiment, the
methods of the present invention are used to prevent or ameliorate
a symptom of anthrax selected from the group consisting of malaise,
fever, dry cough, myalgias, and chest pains, ventilatory
compromise, sweating, widening of the mediastimum on radiographic
studies, edema of the neck and chest, necrotizing mediastinal
lymphadenitis, non-pitting edema, eschar, nausea, vomiting, fever,
abdominal pain, bloody diarrhea, mucosal ulcerations, hemorrhagic
mesenteric lymphadenitis, or any combination thereof.
[0048] In yet another aspect, the present invention is directed to
a method of relieving or ameliorating the pain or symptoms
associated with any one or more of the above-identified bacterial
diseases or indications, mycobacterial diseases or indications, or
anthrax infection in a mammal suffering from any one or more of the
above-identified bacterial diseases or indications, mycobacterial
diseases or indications, or anthrax infection which comprises
administering to the mammal in need thereof a therapeutically
effective pain or symptom-reducing amount of a pharmaceutical
composition comprising effective amounts of a substance exhibiting
mammalian .alpha.1-antitrypsin or inhibitor of serine protease
activity, either alone or in combination with one or more
anti-inflammatory compounds or immunomodulatory agents; and a
pharmaceutically acceptable carrier or excipient, wherein said
mammalian .alpha.1-anitrypsin or inhibitor of serine protease
activity substance is sufficient to inhibit or ameliorate the
bacterial disease or indication, mycobacterial disease or
indication, or anthrax infection of the host.
[0049] In one embodiment, the reduction or inhibition of pain
and/or symptoms associated with one or more of each of the
above-recited mycobacterial indications, bacterial infections or
anthrax infections is on the order of about 10-20% reduction or
inhibition. In another embodiment, the reduction or inhibition of
pain is on the order of 30-40%. In another embodiment, the
reduction or inhibition of pain is on the order of 50-60%. In yet
another embodiment, the reduction or inhibition of the pain
associated with each of the recited indications is on the order of
75-100%. It is intended herein that the ranges recited also include
all those specific percentage amounts between the recited range.
For example, the range of about 75 to 100% also encompasses 76 to
99%, 77 to 98%, etc, without actually reciting each specific range
therewith.
[0050] Accordingly, the overall aspect of the present invention to
provide compounds that exhibit inhibitory activity toward serine
proteases. Thus, it should be recognized that this invention is
applicable to the control of catalytic activity of serine proteases
in any appropriate situation including, but not necessarily limited
to, medicine, biology, agriculture, and microbial fermentation.
[0051] One aspect of the present invention is to provide clinically
acceptable serine protease inhibitors with recognized utility and
exhibiting relatively high activity at relatively low
concentrations.
[0052] In one embodiment, the .alpha.1-antitrypsin used in the
methods and compositions of the present invention comprises
Aralast.TM. (Baxter), Zemaira.TM. (Aventis Behring), Prolastin.RTM.
(Bayer), Aprotonin.TM. or Trasylol.TM. (Bayer Pharmaceutical
Corporation) and Ulinistatin.TM. (Ono Pharmaceuticals, Inc.), or
any combination thereof.
[0053] The present invention provides methods for therapeutically
or prophylactically treating bacterial infections in a subject.
[0054] The method for therapeutically treating bacterial or
mycobacterial infections comprises the step of administering
pharmaceutically effective amounts of a substance exhibiting
mammalian .alpha.1-antitrypsin or inhibitor of serine protease
activity or derivatrive thereof to the subject after occurrence of
the bacteral or mycobacterial disease.
[0055] The method for prophylactically treating bacterial or
mycobacterial infections comprises the step of administering
pharmaceutically effective amounts of a substance exhibiting
mammalian .alpha.1-antitrypsin or inhibitor of serine protease
activity or derivatrive thereof to the subject prior to the
occurrence of the bacterial or mycobacterial disease.
[0056] Either methodology inhibits the bacterial infection or the
mycobacterial infection of macrophages.
[0057] For each of the above-recited methods of the present
invention, the therapeutically effective amount of one or more
substances exhibiting mammalian .alpha.1-antitrypsin or inhibitor
of serine protease activity or a functional derivative thereof may
be administered to a subject in need thereof in conjunction with a
therapeutically effective amount of one or more anti-microbacterial
drugs and/or inflammatory compounds and/or a therapeutically
effective amount of one or more immunomodulatory agents.
[0058] In certain embodiments of the method of the present
invention, the antiinflammatory compound or immunomodulatory drug
comprises interferon; interferon derivatives comprising betaseron,
.beta.-interferon; prostane derivatives comprising iloprost,
cicaprost; glucocorticoids comprising cortisol, prednisolone,
methylprednisolone, dexamethasone; immunsuppressives comprising
cyclosporine A, FK-506, methoxsalene, thalidomide, sulfasalazine,
azathioprine, methotrexate; lipoxygenase inhibitors comprising
zileutone, MK-886, WY-50295, SC-45662, SC-41661A, BI-L-357;
leukotriene antagonists; peptide derivatives comprising ACTH and
analogs thereof; soluble TNF-receptors; TNF-antibodies; soluble
receptors of interleukines, other cytokines, T-cell-proteins;
antibodies against receptors of interleukines, other cytokines,
T-cell-proteins; and calcipotriols and analogues thereof taken
either alone or in combination.
[0059] The present invention also relates to the combined use of
the pharmaceutical composition exhibiting mammalian
.alpha.1-antitrypsin or inhibitor of serine protease activity in
combination with one or more antibacterial or antiviral
compositions or any combination thereof for treating any one of the
aforementioned bacterial or mycobacterial diseases, or any
combination thereof.
[0060] In each of the above-recited methods, the mammalian
.alpha.1-antitrypsin or inhibitor of serine protease activity
substance may be part of a fusion polypeptide, wherein said fusion
polypeptide comprises mammalian .alpha.1-antitrypsin or a substance
with inhibitor of serine protease activity and an amino acid
sequence heterologous to said mammalian .alpha.1-antitrypsin or
inhibitor of serine protease activity substance.
[0061] In certain embodiments, the fusion polypeptide contemplated
for use in the methods of the present invention comprise a human
immunoglobin constant region, such as for example, a human IgG1
constant region, including a modified human IgG1 constant region
wherein the IgG1 constant region does not bind the Fc receptor
and/or does not initiate antibody-dependent cellular cytotoxicity
(ADCC) reactions.
[0062] In yet other embodiments, the fusion polypeptide
contemplated for use in the methods of the present invention can
additionally comprise an amino acid sequence that is useful for
identifying, tracking or purifying the fusion polypeptide, e.g.,
the fusion polypeptide can further comprise a FLAG or HIS tag
sequence. The fusion polypeptide can additionally further comprise
a proteolytic cleavage site which can be used to remove the
heterologous amino acid sequence from the mammalian
.alpha.1-antitrypsin or the substance with inhibitor of serine
protease activity. In each of the above-recited compositions and
methods of the invention the agent that inhibits the bacterial
infection, mycobacterial infection of human
monocyte-derived-macrophages or anthrax comprises
.alpha.1-antitrypsin. In addition, peptides of interest are
homologous and analogous peptides. While homologues are natural
peptides with sequence homology, analogues will be peptidyl
derivatives, e.g., aldehyde or ketone derivatives of such peptides.
Typical examples of analogues are TLCK or TPCK. Without limiting to
.alpha.1-antitrypsin and peptide derivatives of
.alpha.1-antitrypsin, compounds like oxadiazole, thiadiazole,
CE-2072, UT-77, and triazole peptoids are preferred.
[0063] In other embodiments, the agent that inhibits the bacterial
infection, the mycobacterial infection of human
monocyte-derived-macropha-ges and/or anthrax can also be an
inhibitor of serine protease activity, an inhibitor of elastase, or
an inhibitor of proteinase-3. The inhibitor of serine protease
activity can include, but is not limited to, small organic
molecules including naturally-occurring, synthetic, and
biosynthetic molecules, small inorganic molecules including
naturally-occurring and synthetic molecules, natural products
including those produced by plants and fungi, peptides, variants of
.alpha.1-antitrypsin, chemically modified peptides, and proteins.
An inhibitor of serine protease activity has the capability of
inhibiting the proteolytic activity of trypsin, elastase,
kallikrein, and/or other serine proteases.
[0064] In one embodiment of the invention, the peptide can be
protected or derivitized in various ways, e.g., N-terminal
acylation, C-terminal amidation, cyclization, etc. In a specific
embodiment, the N-terminus of the peptide is acetylated.
[0065] The peptides of interest are homologous and analogous
peptides. While homologues are natural peptides with sequence
homology, analogues will be peptidyl derivatives, e.g., aldehyde or
ketone derivatives of such peptides. Without limiting to AAT and
peptide derivatives of AAT, the compounds like oxadiazole,
thiadiazole and triazole peptoids and substances comprising certain
phenylenedialkanoate esters are preferred.
[0066] In each of the above-recited methods, the mammalian
.alpha.1-antitrypsin or inhibitor of serine protease activity
substance contemplated for use within the methods of the present
invention further comprises a series of peptides comprising
carboxyterminal amino acid peptides corresponding to AAT. These
pentapeptides can be represented by a general formula (I):
I-A-B-C-D-E-F-G-H-II, wherein I is Cys or absent; A is Ala, Gly;
Val or absent; B is Ala, Gly, Val, Ser or absent; C is Ser, Thr or
absent; D is Ser, Thr, Ans, Glu, Arg, Ile, Leu or absent; E is Ser,
Thr, Asp or absent; F is Thr, Ser, Asn, Gln, Lys, Trp or absent; G
is Tyr or absent; H is Thr, Gly, Met, Met(O), Cys, Thr or Gly; and
II is Cys, an amide group, substituted amide group, an ester group
or absent, wherein the peptides comprise at least 4 amino acids and
physiologically acceptable salts thereof. Among this series of
peptides, several are equally acceptable including FVFLM (SEQUENCE
ID NO. 1), FVFAM (SEQUENCE ID NO. 2), FVALM (SEQUENCE ID NO. 3),
FVFLA (SEQUENCE ID NO. 4), FLVFI (SEQUENCE ID NO. 5), FLMII
(SEQUENCE ID NO. 6), FLFVL (SEQUENCE ID NO. 7), FLFVV (SEQUENCE ID
NO. 8), FLFLI (SEQUENCE ID NO. 9), FLFFI (SEQUENCE ID NO. 10),
FLMFI (SEQUENCE ID NO. 11), FMLLI (SEQUENCE ID NO. 12), FIIMI
(SEQUENCE ID NO. 13), FLFCI (SEQUENCE ID NO. 14), FLFAV (SEQUENCE
ID NO. 15), FVYLI (SEQUENCE ID NO. 16), FAFLM (SEQUENCE ID NO. 17),
AVFLM (SEQUENCE ID NO. 18), and any combination thereof.
[0067] In yet another embodiment, these peptides can be represented
by a general formula (II): NT-X1-X2-X3-X4-X5-CT or a
physiologically acceptable salt thereof, in which NT comprises an
amino acid residue positioned at the peptide's N-terminal end,
including C, an acetyl group, or a succinyl group, provided that NT
can also be absent; X1 comprises an amino acid residue, including F
or A; X2 comprises an amino acid residue, including C, V, L, M, I,
A, C, or S; X3 comprises an amino acid residue, including F, A, V,
M, L, I, Y, or C; X4 comprises an amino acid residue, including L,
A, F, I, V, M, C, G, or S; X5 comprises an amino acid residue,
including M, A, I, L, V, F, or G; and CT comprises an amino acid
residue positioned at the peptide's C-terminal end, including C, an
amide group, a substituted amide group, or an ester group, provided
that CT can also be absent, and in which the amino acid residue can
be either an L- or a D-stereoisomeric configuration. These peptides
comprise at least 5 amino acids and physiologically acceptable
salts thereof. Amino acids in the formula are abbreviated as
1-letter and corresponding 3-letter codes are as follow: Alanine is
A or Ala; Arginine R or Arg, Asparagine N or Asn; Aspartic acid Dor
Asp; Cysteine C or Cys; Glutamine Q or Gln; Glutamic acid E or Glu;
Glycine G or Gly; Histidine H or His; Isoleucine I or Ile; Leucine
L or Leu; Lysine K or Lys; Methionine M or Met; Phenylalanine F or
Phe; Proline P or Pro; Serine S or Ser; Threonine T or Thr;
Tryptophan W or Tip; Tyrosine Y or Tyr; and Valine V or Val.
[0068] In each of the above-recited methods, the mammalian
.alpha.1-antitrypsin or inhibitor of serine protease activity
substance contemplated for use within the methods of the present
invention further comprises a series of peptides comprising amino
acid peptides corresponding to portions or fragments of AAT. For
example, and not by way of limitation, amino acid peptides
corresponding to 10 amino acid fragments of AAT are specifically
contemplated for use in the composition and methods of the present
invention. In particular, amino acid peptides MPSSVSWGIL (SEQUENCE
ID NO. 19); LAGLCCLVPV (SEQUENCE ID NO. 20) SLAEDPQGDA (SEQUENCE ID
NO. 21); AQKTDTSHHD (SEQUENCE ID NO. 22) QDHPTFNKIT (SEQUENCE ID
NO. 23); PNLAEFAFSL (SEQUENCE ID NO. 24); YRQLAHQSNS (SEQUENCE ID
NO. 25); TNIFFSPVSI (SEQUENCE ID NO. 26); ATAFAMLSLG (SEQUENCE ID
NO. 27); TKADTHDEIL (SEQUENCE ID NO. 28); EGLNFNLTEI (SEQUENCE ID
NO. 29); PEAQIHEGFQ (SEQUENCE ID) NO. 30); ELLRTLNQPD (SEQUENCE ID
NO. 31); SQLQLTTGNG (SEQUENCE ID NO. 32); LFLSEGLKLV (SEQUENCE ID
NO. 33); DKFLEDVKKL (SEQUENCE ID NO. 34); YHSEAFTVNF (SEQUENCE ID
NO. 35); GDHEEAKKQI (SEQUENCE ID NO. 36); NDYVEKGTQG (SEQUENCE ID
NO. 37); KIVDLVKELD (SEQUENCE ID NO. 38); RDTVFALVNY (SEQUENCE ID
NO. 39); IFFKGKWERP (SEQUENCE ID NO. 40); FEVKDTEDED (SEQUENCE ID
NO. 41); FHVDQVTTVK (SEQUENCE ID NO. 42); VPMMKRLGMF (SEQUENCE ID
NO. 43); NIQHCKKLSS (SEQUENCE ID NO. 44); WVLLMKYLGN (SEQUENCE ID
NO. 45); ATAIFFLPDE (SEQUENCE ID NO. 46); GKLQHLENEL (SEQUENCE ID
NO. 47); THDIITKFLE (SEQUENCE ED NO. 48); NEDRRSASLH (SEQUENCE ID
NO. 49); LPKLSITGTY (SEQUENCE ID NO. 50); DLKSVLGQLG (SEQUENCE ID
NO. 51); ITKVFSNGAD (SEQUENCE ID NO. 52); LSGVTEEAPL (SEQUENCE ID
NO. 53); KLSKAVHKAV (SEQUENCE ID NO. 54); LTIDEKGTEA (SEQUENCE ID
NO. 55); AGAMFLEAIP (SEQUENCE ID NO. 56); MSIPPEVKFN (SEQUENCE ID
NO. 57); KPFVFLMIEQ (SEQUENCE ID NO. 58); NTKSPLFMGK (SEQUENCE ID
NO. 59); VVNPTQK (SEQUENCE ID NO. 60), or any combination
thereof.
[0069] It is specifically intended that the AAT peptides recited
contemplated for use in the compositions and methods of the present
invention are also intended to include any and all of those
specific AAT peptides other than the 10 amino acid AAT peptides of
SEQ ID NO. 61 depicted supra. For example, while AAT peptides amino
acids 1-10, amino acids 11-20, amino acids 21-30, etc of SEQ ID NO.
61 have been enumerated herein, it is intended that the scope of
the compositions and methods of use of same specifically include
all of the possible combinations of AAT peptides such as amino
acids 2-12, amino acid 3-13, 4-14, etc. of SEQ ID NO. 61, as well
as any and all AAT peptide fragments corresponding to select amino
acids of SEQ ID NO. 61, without actually reciting each specific AAT
peptide of SEQ ID NO. 61 therewith. Thus, by way of illustration,
and not by way of limitation, Applicants are herein entitled to
possession of compositions based upon any and all AAT peptide
variants based upon the amino acid sequence depicted in SEQ ID NO.
61 and use of such compositions in the methods of the present
invention.
[0070] The AAT and similarly active compounds contemplated for use
in the compositions and methods of the present invention may be
identified by a series of assays wherein a compound (AAT) will
exhibit inhibitory activity versus control in an assay. One of
these assays comprises blocking infection of human monocyte derived
macrophages in an in vitro model of infection as described in
detail in Example 1 of the detailed description of this
disclosure.
[0071] [In one embodiment, with respect to the use of the
compositions and methods of the present invention to prevent or
ameliorate a symptom caused by either Bacillus anthracis,
Corynebacterium diptheriae, or Pseudomonas aeruginosa, specifically
excluded within the scope of the present invention are those furin
endoprotease inhibitors comprising an .alpha.1-antitrypsin variant
having an amino acid sequence comprising the amino acids of the
native .alpha.1-antitrypsin molecule, except that the sequence at
position 355-358 of the native protein (-Ala-Ile-Pro-Met-) is
changed to the novel sequence -Arg-X-X-Arg-, wherein X is any amino
acid, at positions 355-358 of the native .alpha.1-antitrypsin amino
acid sequence as disclosed in U.S. Pat. Nos. 5,604,201 and
6,022,855.
[0072] Also specifically excluded within the scope of the
compositions and methods of the present invention to prevent or
ameliorate a symptom caused by either Bacillus anthracis,
Corynebacterium diptheriae, or Pseudomonas aeruginosa are those
.alpha.1-antitrypsin Portland variants wherein the amino acid
sequence at positions 355-358 of the .alpha.1-antitrypsin amino
acid Portland sequence is -Arg-Ile-Pro-Arg- as disclosed in U.S.
Pat. Nos. 5,604,201 and 6,022,855.
[0073] Also specifically excluded within the scope of the
compositions and methods of the present invention to prevent or
ameliorate a symptom caused by either Bacillus anthracis,
Corynebacterium diptheriae, or Pseudomonas aeruginosa are peptides
having amino acid sequences of about 4 to about 100 amino acids in
length comprising the amino acid sequence -Arg-Xaa-Xaa-Arg-,
wherein each Xaa is any amino acid as is disclosed in U.S. Pat.
Nos. 5,604,201 and 6,022,855.
[0074] In yet another embodiment, with respect to the use of the
compositions and methods of the present invention to prevent or
ameliorate a symptom of anthrax, specifically excluded within the
scope of the present invention are those furin endoprotease
inhibitors comprising HexArg as disclosed in Miroslav S. Sarac et
al. (Infection and Immunity, January 2004, p. 602-605, Vol. 72, No.
1 Protection against Anthrax Toxemia by Hexa-D-Arginine In Vitro
and In Vivo).
[0075] The invention further provides pharmaceutical compositions
comprising such agents.
[0076] The preferred doses for administration can be anywhere in a
range between about 10 ng and about 10 mg per ml or mg of the
formulation. The therapeutically effective amount of AAT peptides
or drugs that have similar activities as AAT or peptides drug can
be also measured in molar concentrations and may range between
about 1 nM and about 10 mM. The formulation is also contemplated in
combination with a pharmaceutically or cosmetically acceptable
carrier. The precise doses can be established by well known routine
clinical trials without undue experimentation.
[0077] In one aspect of the invention, the pharmaceutical
compositions of the present invention are administered orally,
systemically, via an implant, intravenously, topically,
intrathecally, intracranially, intraventricularly, by inhalation or
nasally.
[0078] In certain embodiments of the methods of the present
invention, the subject or mammal is a human.
[0079] In other embodiments of the methods of the present
invention, the subject or mammal is a veterinary and/or a
domesticated mammal.
[0080] There has been thus outlined, rather broadly, the important
features of the invention in order that a detailed description
thereof that follows can be better understood, and in order that
the present contribution can be better appreciated. There are
additional features of the invention that will be described
hereinafter.
[0081] In this respect, before explaining at least one embodiment
of the invention in detail, it is to be understood that the
invention is not limited in its application to the details as set
forth in the following description and figures. The present
invention is capable of other embodiments and of being practiced
and carried out in various ways. Additionally, it is to be
understood that the terminology and phraseology employed herein are
for the purpose of description and should not be regarded as
limiting.
[0082] As such, those skilled in the art will appreciate that the
conception, upon which this disclosure is based, can readily be
used as a basis for designing other methods for carrying out the
several features and advantages of the present invention. It is
important, therefore, that the claims be regarded as including such
equivalent constructions insofar as they do not depart from the
spirit and scope of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0083] FIG. 1 illustrates the effect of .alpha.-1-antitrypsin (AAT)
and AAT mimic on mycobacterium avium complex (mac) infection of
human monocyte-derived macrophages (n=4).
[0084] FIG. 2 illustrates the effect of .alpha.-1-antitrypsin (AAT)
and AAT mimic on mycobacterium avium complex (mac)-induced
TNF.alpha. in human monocyte-derived macrophages.
[0085] FIG. 3 illustrates the effect of .alpha.-1-antitrypsin (AAT)
and aat mimic on mycobacterium avium complex (mac)-induced
TNF.alpha. in human monocyte-derived macrophages: time-course
experiment (n=1).
[0086] FIGS. 4A-4H illustrate the bacillus anthracis toxin
mechanism and the method by which serine protease inhibitors
neutralize the toxin.
[0087] FIG. 5 illustrates the effect of .alpha.-1-antitrypsin on
stimulated interleukin-1 beta production in whole human blood.
DETAILED DESCRIPTION OF THE INVENTION
Standard Methods
[0088] In accordance with the present invention there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory
Manual, Second Edition 1989, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y.; Animal Cell Culture, R. I. Freshney, ed.,
1986).
Therapeutic Methods
[0089] The present invention provides methods for treating
mycobacterial infections comprising administering to a subject in
need thereof of a therapeutically effective amount of a composition
comprising an effective amount of a substance exhibiting mammalian
.alpha.1-antitrypsin or inhibitor of serine protease activity or a
functional derivative thereof; and a pharmaceutically acceptible
excipient.
[0090] According to the methods of the present invention,
mycobacterial infection of macrophages is inhibited to obtain
important therapeutic benefits.
[0091] Therefore, administration of a dosage of the invention
composition, i.e., .alpha.1-antitrypsin, or a fragment, derivative
or analog thereof, can be beneficial for the treatment of
mycobacterial diseases or disorders. In a preferred aspect, the
agent is an analog of .alpha.1-antitrypsin that can cross the blood
brain barrier, which would allow for intravenous or oral
administration. Many strategies are available for crossing the
blood brain barrier, including but not limited to, increasing the
hydrophobic nature of a molecule; introducing the molecule as a
conjugate to a carrier, such as transferrin, targeted to a receptor
in the blood brain barrier; and the like. In another embodiment,
the agent can be administered intracranially or, more directly,
intraventricularly. In yet another embodiment, the agent can be
administered by way of inhalation or nasally.
[0092] In a further embodiment, the methods and compositions of the
invention are useful in the therapeutic treatment of mycobacterial
diseases or disorders of the immune system. In a yet further
embodiment, diseases can be prevented by the timely administration
of the agent of the invention as a prophylactic, prior to onset of
symptoms, or signs, or prior to onset of severe symptoms or signs
of a mycobacterial disease. Thus, a patient at risk for a
particular mycobacterial disease can be treated with serine
protease inhibitors, for example,
(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(3-Trifluoromethylbenzyl)-1,2,4-ox-
adiazolyl)carbonyl)-2-(S)-Methylpropyl]-L-Prolinamide; as a
precautionary measure.
[0093] The effective dose of the agent of the invention, and the
appropriate treatment regime, can vary with the indication and
patient condition, and the nature of the molecule itself, e.g., its
in vivo half life and level of activity. These parameters are
readily addressed by one of ordinary skill in the art and can be
determined by routine experimentation.
[0094] The preferred doses for administration can be anywhere in a
range between about 0.01 mg and about 20 mg per ml of biologic
fluid of treated patient. The therapeutically effective amount of
.alpha.1-antitrypsin, peptides, or drugs that have similar
activities as .alpha.1-antitrypsin or peptides can be also measured
in molar concentrations and can range between about 1 nM to about 2
mM.
Serine Protease Inhibitors
[0095] It is to be understood that the present invention is not
limited to the examples described herein, and other serine
proteases known in the art can be used within the limitations of
the invention. For example, one skilled in the art can easily adopt
inhibitors as described in WO 98/24806, which discloses substituted
oxadiazole, thiadiazole and triazole as serine protease inhibitors.
U.S. Pat. No. 5,874,585 discloses substituted heterocyclic
compounds useful as inhibitors of serine proteases; including:
(benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(3-trifluoro-methylbenzyl)-1,2,4-o-
xa
diazolyl)carbonyl)-2-(S)-methylpropyl]-L-prolinami-debenzyloxycarbonyl)-
-L-valyl-N-[1-(3-(5-(2-phenylethyl)-1,2,4-oxadiazolyl-)carbonyl)-2-(S)-met-
hylpropyl]-L-prolinamide;
(benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(2-methoxybenzyl)-1,2,4-oxadiazoly-
l)carbonyl)-2-(S)-methylpropy-1]-L-prolinamide;
(benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(trifluoromethyl)-1-1,2,4-oxadiazo-
lyl)carbonyl)-2-(S)-methylpropyl]-L-prolinamide;
(benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(methyl)-1,2,4-oxadiazolyl)carbony-
l)-2-(S)-Methylpropyl]-L-Prolinamide;
(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(difluoromethyl)-1,2,4-oxadiazolyl-
)carbonyl)-2-(S)-Methylpropyl]-L-Prol-inamide;
(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(benzyl)-1,2,4-oxadiazolyl-)carbon-
yl)-2-(S)-Methylpropyl]-L-Prolinamide;
(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(3-methoxybenzyl)-1,2,4-oxadiazoly-
l)carbonyl)-2-(S)-Methylpropy-1]-L-Prolinamide;
(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(2,6-difluorobenzyl)-1,2,4-oxadiaz-
olyl)carbonyl)-2-(S)-Methylpropyl]-L-Prolinamide;
(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(trans-styryl)-1,2,4-oxadiazolyl)c-
arbonyl)-2-(S)-Methylpropyl]-L-Prolinamide;
(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(trans-4-Trifluoro
methylstyryl)-1,2,4-oxadiazolyl)carbonyl)-2-(S-)-Methylpropyl]-L-Prolinam-
ide;
(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(tra-ns-4-Methoxystyryl)-1,2,4-
-oxadiazolyl)carbonyl)-2-(S)-Methylpropyl]-L-Prol-inamide;
(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(3-Thienylmethyl)-1,2,4-oxadiazoly-
l)carbonyl)-2-(S)-Methylpropyl]-L-Prolinamide;
(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(Phenyl)-1,2,4-oxadiazolyl)carbony-
l)-2-(S)-methylpropyl]-L-prolinamide; and
(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(3-Phenylpropyl)-1,2,4-oxadiazolyl-
)carbonyl)-2-(S)-Methylpropyl]-L-Prolinamide. U.S. Pat. No.
5,216,022 teaches other small molecules useful for the practice of
this invention, including:
Benzyloxycarbonyl-L-valyl-N-[1-(2-[5-(3-methylbenzyl)-1,3,4-oxadiazolyl]c-
arbonyl)-2-(S)-methylpropyl-]-L-prolinamide (also known as
CE-2072),
Benzyloxycarbonyl-L-valyl-N-[1-(2-(3-methylbenzyl)-1,3,4-oxadiazolyl]carb-
onyl)-2-(S)-methylpropyl]-L-prolin-amide;
Benzyloxycarbonyl-L-valyl-N-[-(2-(5-(methyl)-1,3,4-oxadiazoly]carbo-nyl)--
2-(S)-methylpropyl]-L-prolinamide;
Benzyloxycarbonyl)-L-valyl-N-[1-(2-(5-(3-trifluoromethylbenzyl)-1,3,4-oxa-
diazolyl]carbonyl)-2-(S)-methylprop-yl]-L-prolinamide;
(Benzyloxycarbonyl)-L-valyl-N-[1-(2-(5-(4-Dimethylamino
benzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide;
Benzyloxycarbonyl)-L-valyl-N-[1-(2-(5-(1-napthylenyl)-1,3,4-oxadiazolyl]c-
-arbonyl)-2-(S)-methylpropyl]-L-prolinamide;
(Benzyloxycarbonyl)-L-valyl-[1-(3-(5-(3,4-methylenedioxybenzyl)-1,2,4-oxa-
diazolyl]carbonyl)-2-(S)-methyl-propyl]-L-prolinamide;
Benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(3,5-dimethyl-benzyl)-1,2,4-oxadiaz-
olyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide;
(Benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(3,5-dimethoxybenzyl)-1,2,4-oxadia-
-zolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide;
(Benzyloxycarbonyl)-L-v-alyl-N-[1-(3-(5-(3,5-ditrifluoromethylbenzyl)-1,2-
,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide;
(Benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(3-methylbenzyl)-1,2,4-oxadiazolyl-
]carbonyl)-2-(S)-methylpropyl]-L-prolina-mide;
(Benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(biphenylmethine)-1,2,4-oxadi-azol-
yl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide;
(Benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(4-phenylbenzyl)-1,2,4-oxadiazolyl-
-]carbonyl)-2-(S)-methylpropyl]-L-prolinamide;
(Benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(3-phenylbenzyl)-1,2,4-oxadiazolyl-
]carbonyl)-2-(S)-methylpropyl-]-L-prolinamide;
(Benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(3-phenoxybenzyl)-1,2,4-oxadiazoly-
l]carbonyl)-2-(S)-methylpropyl]-L-prolinamide;
(Benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(cyclohexylmethylene)-1,2,4-oxadia-
-zolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide;
(Benzyloxycarbonyl)-L-v-alyl-N-[1-(3-(5-(3-trifluoromethyldimethylmethyle-
ne)-1,2,4-oxadiazolyl]car-bonyl)-2-(S)-methylpropyl]-L-prolinamide;
(Benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(1-napthylmethylene)-1,2,4-oxadiaz-
olyl]carbonyl)-2-(S)-methylpropyl-]-L-prolinamide;
(Benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(3-pyridylmethyl)-1,2,4-oxadiazoly-
l]carbonyl)-2-(S)-methylpropyl]-L-prolinamide;
(Benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(3,5-diphenylbenzyl)-1,2,4-oxadiaz-
-olyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide;
(Benzyloxycarbonyl)-L-va-lyl-N-[1-(3-(5-(4-dimethylaminobenzyl)-1,2,4-oxa-
diazolyl]carbonyl)-2-(S)-m-ethylpropyl]-L-prolinamide;
2-(5-[(Benzyloxycarbonyl)amino]-6-oxo-2-(4-flu-orophenyl)-1,6-dihydro-1-p-
yrimidinyl]-N-[1-(3-(5-(3-trifluoromethylbenzyl)-1,2,4-oxadiazolyl]carbony-
l)-(S)-2-methylpropyl]acetamide;
2-(5-Amino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(3-(5-
-(3-trifluoromethylbenzyl)-1,2,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-
-acetamide;
2-(5-[(Benzyloxycarbonyl)amino]-6-oxo-2-(4-fluorophenyl)-1,6-di-hydro-1-p-
yrimidinyl]-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbony-l)-(S)-2-
-methylpropyl]acetamide;
2-(5-Amino-6-oxo-2-(4-fluorophenyl)-1,6-d-ihydro-1-pyrimidinyl]-N-[1-(2-(-
5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbon-yl)-2-methylpropyl]acetamide;
(Pyrrole-2-carbonyl)-N-(benzyl)glycyl-N-[1-(-2-(5-(3-methylbenzyl)-1,3,4--
oxadiazolyl]carbonyl)-2-(S)-methylpropyl]amide-;
(Pyrrole-2-carbonyl)-N-(benzyl)glycyl-N-[1-(3-(5-(3-trifluoromethylbenzy--
l)]-1,2,4-oxadiazolyl)-(S)-methylpropyl]amide;
(2S,5S)-5-Amino-1,2,4,5,6,7-hexahydroazepino-[3,2,1]-indole-4-one-carbony-
l-N-[1-(2-(5-(3-methylbenzyl-)-1,3,4-oxadiazolyl]carbonyl)-(R,S)-2-methylp-
ropyl]amide;
BTD-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpro-
-pyl]amide;
(R,S)-3-Amino-2-oxo-5-phenyl-1,4,-benzodiazepine-N-[1-(2-(5-(3-methylbenz-
yl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;
(Benzyloxycarbonyl)-L-valyl-2-L-(2,3-dihydro-1H-indole)-N-[1-(2-(5-(3-met-
-hylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]amide;
(Benzyloxycarbonyl)-L-valyl-2-L-(2,3-dihydro-1H-indole)-N-[1-(3-(5-(3-tri-
-fluoromethylbenzyl)-1,2,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]amide;
Acetyl-2-L-(2,3-dihydro-1H-indole)-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxad-
-iazolyl]carbonyl)-2-(S)-methylpropyl]amide;
3-(S)-(Benzyloxycarbonyl)amino-)-.epsilon.-lactam-N-[1-(2-(5-(3-methylben-
zyl)-1,3,4-oxadiazolyl]carbonyl-)-2-(S)-methylpropyl]acetamide;
3-(S)-(Amino)-.epsilon.-lactam-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazo-
lyl]carbonyl)-2-(S)-methylpropyl]acetamide trifluoroacetic acid
salt; 3-(S)-[(4-morpholino
carbonyl-butanoyl)amino]-.epsilon.-lactam-N-[1-(2-(5-(3-methylbenzyl)-1,3-
,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide;
6-[4-Fluorophenyl]-.epsilon.-lactam-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxa-
diazolyl]carbonyl)-2-(S)-methylpropyl]acetam-ide;
2-(2-(R,S)-Phenyl-4-oxothiazolidin-3-yl]-N-[1-(2-(5-(3-methylbenzyl)-1,3,-
4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;
2-(2-(R,S)-phenyl-4-oxothiazolidin-3-yl]-N-[1-(2-(5-(3-methylbenzyl)-1,3,-
-4-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]acetamide;
2-(2-(R,S)-Benzyl-4-oxothiazolidin-3-yl]-N-[1-(2-(5-(3-methylbenzyl)-1,3,-
-4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-acetamide;
2-(2-(R,S)-Benzyl-4-oxothiazolidin-3-yl
oxide]-N-[1-(3-(5-(3-trifluorometh-ylbenzyl)-1,2,4-oxadiazolyl]carbonyl)--
2-(R,S,)-methylpropyl]acetamide;
(1-Benzoyl-3,8-quinazolinedione)-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadia-
-zolyl]carbonyl)-2-(S)-methylpropyl]acetamide;
(1-Benzoyl-3,6-piperazinedio-ne)-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadia-
zolyl]carbonyl)-2-(S)-methylpr-opyl]acetamide;
(1-Phenyl-3,6-piperazinedione)-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazo-
lyl]carbonyl)-2-(S)-methylpropyl]acetamide;
[(1-Phenyl-3,6-piperazinedione)-N-[1-(3-(5-(3-trifluoromethylbenzyl)-1,2,-
-4-oxadiazolyl]carbonyl)]-2-(S)-methylpropyl]acetamide;
3-[(Benzyloxycarbonyl)amino]-quinolin-2-one-N-[1-(2-(5-(3-methylbenzyl)-1-
-,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;
3-[(Benzyloxycarbonyl)amino]-7-piperidinyl-quinolin-2-one-N-[1-(2-(5-(3-m-
-ethylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;
3-(Carbomethoxy-quinolin-2-one-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazo-
l-yl]carbonyl)-2-(S)-methylpropyl]acetamide;
3-(Amino-quinolin-2-one)-N-[1-(-2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]c-
ar bonyl)-2-(S)-methylpropyl]acet-amide;
3-[(4-Morpholino)aceto]amino-quinolin-2-one-N-[1-(2-(5-(3-methylben-zyl)--
1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;
3,4-Dihydro-quinolin-2-one-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]-
-carbonyl)-2-(S)-methylpropyl]acetamide;
1-Acetyl-3-(4-fluorobenzylidene)p-piperazine-2,5-dione-N-[1-(2-(5-(3-meth-
ylbenzyl)-1,3,4-oxadiazolyl]carbonyl-)-2-(S)-methylpropyl]acetamide;
1-Acetyl-3-(4-dimethylamino
benzylidene)piperazine-2,5-dione-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadia-
-zolyl]carbonyl)-2-(S)-methylpropyl]acetamide;
1-Acetyl-3-(4-carbomethoxy
benzylidene)piperazine-2,5-dione-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadia-
-zolyl]carbonyl)-2-(S)-methylpropyl]acetamide;
1-Acetyl-3-[(4-pyridyl)methyl-lene]piperazine-2,5-dione-N-[1-(2-(5-(3-met-
hyl
benzyl)-1,3,4-oxadiazolyl]c-arbonyl)-2-(S)-methylpropyl]acetamide;
4-[1-Benzyl-3-(R)-benzyl-piperazine-1-2,5,-dione]-N-[1-(2-[5-(3-methylben-
zyl)-1,3,4-oxadiazolyl]carbonyl)-2-met-hylpropyl]acetamide;
4-[1-Benzyl-3-(S)-benzyl
piperazine-2,5,-dione]-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxadiazolyl]carb-
onyl)-2-(S)-methylpropyl]acet-amide;
4-[1-Benzyl-3(R)-benzylpiperazine-2,5,-dione]-N-[1-(3-(5-(3-trifluo-romet-
hylbenzyl)-1,2,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;
4-[1-Benzyl-3-(S)-benzylpiperazine-2,5,-dione]-N-[1-(3-(5-(3-trifluoromet-
-hylbenzyl)-1,2,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;
4-[1-Benzyl-3-(S)-benzyl
piperazine-2,5,-dione]-N-[1-(3-(5-(2-dimethylami-noethyl)-1,2,4-oxadiazol-
yl]carbonyl)-2-(S)-methylpropyl]acetamide;
4-[1-Methyl-3-(R,S)-phenylpiperazine-2,5,-dione]-N-[1-(3-(5-(3-trifluorom-
-ethylbenzyl)-1,2,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;
4-[1-Methyl-3-(R,S)-phenyl
piperazine-2,5,-dione]-N-[1-(2-(5-(3-methylben-zyl)-1,3,4-oxadiazolyl]car-
bonyl)-2-(S)-methylpropyl]acetamide; 4-[1-(4-Morpholino
ethyl)3-(R)-benzyl
piperazine-2,5,-dione]-N-[1-(2-(5-(-3-methylbenzyl)-1,3,4-oxadiazolyl]car-
bonyl)-2-(S)-methylpropyl]acetamide;
5-(R,S)-Phenyl-2,4-imidazolidinedione-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-o-
-xadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;
5-(R)-Benzyl-2,4-imidaz-olidinedione-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-ox-
adiazolyl]carbonyl)-2-(S-methylpropyl]acetamide;
5-(S)-Benzyl-2,4-imidazolidinedione-N-[1-(2-(5-(3-methylbenzyl)-1,3,4-oxa-
diazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;
5-(S)-Benzyl-2,4-imidazolidinedione-N-[1-(3-(5-(3-trifluoromethylbenzyl)--
1,2,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;
5-(R)-Benzyl-2,4-imidazolidinedione-N-[1-(3-(5-(3-trifluoromethylbenzyl)--
1,2,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide;
1-Benzyl-4-(R)-benzyl-2,5-imidazolidinedione-N-[1-(2-(5-(3-methylbenzyl)--
1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide; and
1-Benzyl-4-(R)-benzyl-2,5-imidazolidinedione-N-[1-(3-(5-(3-trifluoromethy-
l-1
benzyl)-1,2,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide
among others.
[0096] Likewise, U.S. Pat. No. 5,869,455 discloses N-substituted
derivatives; U.S. Pat. No. 5,861,380 protease inhibitors-keto and
di-keto containing ring systems; U.S. Pat. No. 5,807,829 serine
protease inhibitor-tripeptoid analogues; U.S. Pat. No. 5,801,148
serine protease inhibitors-proline analogues; U.S. Pat. No.
5,618,792 substituted heterocyclic compounds useful as inhibitors
of serine proteases. These patents and PCT publications and others
as listed infra are incorporated herein, in their entirety, by
reference. Other equally advantageous molecules, which may be used
instead of .alpha.1-antitrypsin or in combination with
.alpha.1-antitrypsin are contemplated such as in WO 98/20034
disclosing serine protease inhibitors from fleas. Without limiting
to this single reference one skilled in the art can easily and
without undue experimentation adopt compounds such as in WO98/23565
which discloses aminoguanidine and alkoxyguanidine compounds useful
for inhibiting serine proteases; WO98/50342 discloses
bis-aminomethylcarbonyl compounds useful for treating cysteine and
serine protease disorders; WO98/50420 cyclic and other amino acid
derivatives useful for thrombin-related diseases; WO 97/21690
D-amino acid containing derivatives; WO 97/10231 ketomethylene
group-containing inhibitors of serine and cysteine proteases; WO
97/03679 phosphorous containing inhibitors of serine and cysteine
proteases; WO 98/21186 benzothiazo and related heterocyclic
inhibitors of serine proteases; WO 98/22619 discloses a combination
of inhibitors binding to P site of serine proteases with chelating
site of divalent cations; WO 98/22098 a composition which inhibits
conversion of pro-enzyme CPP32 subfamily including caspase 3
(CPP32/Yama/Apopain); WO 97/48706 pyrrolo-pyrazine-diones; WO
97/33996 human placental bikunin (recombinant) as serine protease
inhibitor; WO 98/46597 complex amino acid containing molecule for
treating viral infections and conditions disclosed hereinabove.
[0097] Other compounds having serine protease inhibitory activity
are equally suitable and effective for use in the methods of the
present invention, including but not limited to: tetrazole
derivatives as disclosed in WO 97/24339; guanidinobenzoic acid
derivatives as disclosed in WO 97/37969 and in a number of U.S.
Pat. Nos. 4,283,418; 4,843,094; 4,310,533; 4,283,418; 4,224,342;
4,021,472; 5,376,655; 5,247,084; and 5,077,428; phenylsulfonylamide
derivatives represented by general formula in WO 97/45402; novel
sulfide, sulfoxide and sulfone derivatives represented by general
formula in WO 97/49679; novel amidino derivatives represented by
general formula in WO 99/41231; other amidinophenol derivatives as
disclosed in U.S. Pat. Nos. 5,432,178; 5,622,984; 5,614,555;
5,514,713; 5,110,602; 5,004,612; and 4,889,723 among many
others.
Mycobacterial Diseases Addressed by the Invention
[0098] Specific mycobacterial diseases or disorders for which the
therapeutic methods of inhibiting the mycobacterial infection of
macrophages of the invention are beneficial include, but are not
limited to, those mycobacterial diseases or disorders caused by
mycobacteria from the genus mycobacterium that includes M.
tuberculosis, M. bovis, M. leprae, M. avium-intracellulare, M.
chelonei (also known as borstelense and abscessus), M. africanum,
M. marinium (also known as balnei and platypoecilus), M. buruli
(also known as ulcerans), M. fortuitum (also known as giae,
minetti, and ranae), M. haemophilum, M. intracellulare, M. kansasii
(also known as luciflavum), M. littorale (also known as xenopi), M.
malmoense, M. marianum (also known as scrofulaceum and
paraffinicum), M. simiae, M. szulgai, M. ulcerans, M. avium (also
known as brunense), M. flavascens, M. lepraemurium, M. microti, and
M. paratuberculosis (which is the causative agent for Johne's
Disease, and a possible cause of Crohn's disease), M. gordonae
(also known as aquae), M. gastri, M. phlei (also known as moelleri
and as timothy bacillus), M. nonchromogenicum, M. smegmatis, M.
terrae, M. triviale, and M. vaccae.
[0099] In another embodiment, the mycobacterium inhibited from
infecting macrophages comprises a mycobacterium from the genus
mycobacterium that includes non-tuberculosis mycobacteria that are
divided into four groups comprising Runyon groups, selected from
the group consisting of Group I (slow-growing photochromogens),
Group II (slow-growing scotochromogens), Group III (slow-growing
nonphotochromogens), and Group IV (rapidly-growing
mycobacteria).
Bacillus anthracis and Anthrax Toxin
[0100] Anthrax toxin, produced by the gram positive rod-shaped
aerobic, spore-forming bacterium Bacillus anthracis, is the toxic
virulence factor secreted by this organism. B. anthraxis is often
considered for use as a biological weapon due to the potency of the
secreted exotoxin, and to the capacity of the bacterium to form
dormant spores which resist harsh environmental conditions.
Sporulation enables ready transport and distribution of large
quantities of toxin-producing bacteria. The toxin is actually a
composite consisting of 3 separate secreted proteins from the
bacterium. The 3 proteins are protective antigen (PA), lethal
factor (LF), and edema factor (EF). While LF and EF directly damage
cells and are thought to cause disease due to anthrax toxin
exposure, the PA is the focus of this present disclosure. PA is
crucial to the virulence of anthrax toxin, since the PA molecule is
designed to import both LF and EF inside the membranes of cells. In
the absence of PA-induced intracellular transport, anthrax toxin is
unable to effect tissue destruction, since LF and EF only function
from within the cell. The importance of PA in the function of
anthrax toxin is underscored by the effective use of PA as the
immunogen in anthrax vaccine. By generating an immune response
against PA, the vaccine confers protection against full (3
component) anthrax toxin.
[0101] A closer examination of the interaction between PA and the
host cells attacked by anthrax toxin is instructive. PA is first
secreted by B. anthracis in a large and functionally inactive form.
This inactive PA binds to a receptor on the surface of host cells.
The PA receptor has recently been isolated and sequenced, and found
to possess von Willebrand Factor-like regions. After docking on the
surface of host cells, PA interacts with a protease present on the
cell surface. The protease cuts (processes) the large and inactive
PA molecule into a smaller and active remnant. The identity of this
protease has been the focus of scant research effort, and it is
poorly characterized. However, prior studies have shown that the
protease has characteristics that suggest it is a host-derived
serine protease. A possible serine protease candidate noted in the
literature is furin (itself a serine protease), but other serine
proteases, such as elastase, proteinase-3, or trypsin are possible
alternatives. Once processed by the action of the cell-surface
serine proteas(s), the activated PA molecules self-assemble into
groups of 7 (heptamers) on the cell surface. These heptames
function as the transport vehicle to deliver LF and EF inside of
the cell. Once inside the cell, LF and EF initiate abnormalities in
cell function.
[0102] A novel approach to nullify the action of anthrax toxin is
to block access of the toxin to the interior of the cell. The
present inventor has shown, in previous extensive laboratory
studies (Leland Shapiro et al. Facet 2000 vol. 15: 115-122, and
unpublished unpublished data of Dr. Leland Shapiro), that serine
proteases residing on the cell surface can be neutralized by the
action of several types of molecules which inhibit serine protease
function. The most important natural, endogenous inhibitor of
serine proteases is .alpha.-1-antitrypsin (AAT). It is noteworthy
that AAT levels are reduced in lymphatics, and that anthrax toxin
production and disease manifestations originate from within the
lymphatics. It is possible that toxin production occurs in
lymphatic tissues because the reduced amounts of AAT provide a
microenvironment conducive to enhanced serine protease function.
Such conditions are expected to augment production of activated
anthrax toxin. Therefore, administering to the subject a
pharmaceutically effective amount of a substance exhibiting
mammalian .alpha.1-antitrypsin or inhibitor of serine protease
activity serves to attenuate or abolish the activity of anthrax
toxin by blocking the activity of the host-derived serine protease
that resides on the cell surface. This will negate the cell-surface
processing of inactive large PA into the active smaller PA remnant.
Thus, by interfering with the host-derived serine protease's
activity, this will disrupt the ability of heptameric PA63 to form
the prepore and ultimately the pore. By disarming the anthrax toxin
using this novel approach (See FIGS. 4A-4H) several advantages are
obtained compared to alternative approaches, for example, and not
by way of limitation:
[0103] 1. Serine protease inhibition, as a strategy to treat
anthrax infection, is highly likely to be impervious to bacterial
mutation due to selective pressure. By choosing to target or
inhibit the serine proteases of host cell origin, the target
molecule is immutable.
[0104] 2. Synthetic inhibitors of serine proteases (AAT-like
mimics) can and have been developed (See, infra, CE-2072). Such a
pharmaceutical agent may be formulated into a pill for oral
consumption in the field or formulated as an inhaler to treat
inhalation anthrax.
[0105] 3. Commercially available agents already approved for
alternate use in humans will work as a treatment for anthrax. These
agents are currently used for indications other than anthrax
toxicity, and include injectible AAT, plasma preparations,
aprotinin and others (American J. Of Resp Critical Care Med 1998,
VII 158: 49-59). One possible instantiation of this invention may
be of immediate practical application. Inhibitors of serine
proteases have been delivered to patients by inhalation. Since the
most lethal form of anthrax infection is pulmonary invasion, an
inhaled agent (natural AAT or a synthetic AAT-like mimic/or other
inhibitor of serine protease) may be especially useful due to
elevated local concentrations, ease of drug delivery, and lack of
side effects (since administration is not systemic). This mode of
focused drug delivery may augment serine protease inhibitor
activity within the pulmonary and mediastinal lymphatics, which are
the principle sites where anthrax is thought to initiate fulminant
disease.
[0106] 4. By neutralizing the anthrax toxin, the direct cause of
disease is disrupted in infected individuals. Antibiotics, on the
other hand, do not target toxin activity, and cannot affect toxin
produced prior to destruction of the bacteria. This invention
specifically contemplates inhibiting host cell serine proteases in
conjunction with administration of one or more anti-bacterial
antibiotics. Antibiotics will stop further toxin production by
preventing the growth of bacteria and/or killing the bacterial
source of toxin.
[0107] 5. This approach to anthrax therapy is likely to be safe.
There is an extensive clinical experience using injectible AAT to
treat patients with genetic AAT deficiency. No long-term untoward
effects have been detected to date (American J. Of Resp Critical
Care Med 1998, VII 158: 49-59; Wencker et al. Chest 2001
119:737-744). Moreover, a small molecule inhibitor of host serine
protease has been administered to patients with Kawasaki's Disease
(Ulinistatin, Ono pharmaceuticals), with an excellent safety and
tolerability record. In addition, inhibition of host serine
proteases to treat anthrax infection will only require a short
treatment course, thus minimizing any potential concerns with long
term exposure to AAT or AAT-like mimics/or other inhibitors of
serine protease.
[0108] 6. Soluble anthrax receptors (Bradley et al. Nature 2001
vol. 414), bacteriophage lysis of anthrax organisms (Schuch et al.
Nature 2002 vol. 418 884-889), dominant negative mutant anthrax
toxin components (Sellman et al. Science 2001 VI. 292: 695-697) and
polyvalent inhibitors (Mourez et al Nature Biotech 2001 Vol. 19,
958-961) may also be used in conjunction with the anthrax-based
methods of the present invention.
[0109] Thus, in view of the above, the present invention provides
methods for preventing a symptom of anthrax in a subject suspected
of having been exposed to or thought to be at risk for exposure to
Bacillus anthracis comprising administering to the subject a
pharmaceutically effective amount of a substance exhibiting
mammalian .alpha.1-antitrypsin or inhibitor of serine protease
activity. The present invention also provides a method for
ameliorating a symptom of anthrax in a subject in need of said
amelioration comprising administering to the subject a
pharmaceutically effective amount of a substance exhibiting
mammalian .alpha.1-antitrypsin or inhibitor of serine protease
activity.
[0110] In each of the above-recited methods, the clinical symptoms
of anthrax can be inhibited or prevented by administration of a
substance exhibiting mammalian .alpha.1-antitrypsin or inhibitor of
serine protease activity.
Clinical Symptoms of Anthrax
[0111] Anthrax occurs as three general clinical entities: i)
inhalation, ii) cutaneous, and iii) gastrointestinal forms.
[0112] i) Inhalation anthrax is the deadliest form of the disease,
and it is the one most likely to be involved in a bioweapons
altercation or accident. Usually, an infected person inhales
anthrax spores serendipitously, or during a bioweapons attack.
Following a 1-6 day incubation period, a biphasic illness ensues.
Initially, there is non-specific malaise/fever/dry cough/myalgias,
and chest pains. The second phase occurs 2-3 days after the first
phase, and consists of progression of the constitutional
non-specific findings listed above, an addition to ventilatory
compromise, sweating, widening of the mediastimum on radiographic
studies, and edema of the neck and chest. This stage of illness is
characterized by a necrotizing mediastinal lymphadenitis. This
second stage of disease can rapidly progress to shock and death
within 2 days, and mortality rates of up to 80% have been reported.
The mechanism of death in animal models appears to be enhanced
production of pro-inflammatory cytokines, especially IL-1. It is of
note, referable to the instant invention disclosure, that lymph
tissue is deficient in serine protease inhibitor activity compared
to other body tissues. The implication is that anthrax toxin is
selectively activated in regions of the body (lymphatics) where
there is an imbalance in serine protease/anti-serine protease
function that favors serine protease activity. A preferred
embodiment for using the instant invention to treat inhalation
anthrax is to deliver large amounts of a serine protease inhibitor
(natural or synthetic) by inhalation. This will result in a shift
in the serine protease/serine protease inhibitor balance in
pulmonary and mediastinal lymphatic tissues toward antiprotease
activity. This will result in blockade of the cell-surface
processing event that is required for activity of anthrax
toxin.
[0113] ii) Cutaneous anthrax is the commonest form (>95%) of
anthrax infection in humans. Upon exposure to anthrax spores,
regions of denuded skin (cuts, abrasions, etc.), present an
environment that enables anthrax organisms to emerge from the spore
state, to grow and replicate, and produce anthrax toxin. Within 1
week, the area of anthrax innoculation develops a painless papule.
Vesicles then form on or near the papule over the ensuing 1-2 days,
followed shortly by development of fever and malaise, and a
non-pitting edema surrounding the skin lesion that is due to toxin
activity. The original lesion (often now a vesicle) ruptures to
form necrotic ulceration and enlargement--this results in formation
of the eschar that characterizes cutaneous anthrax infection. In
the absence of therapy, this disease carries a 20% morality. For
those who recover, the eschar sloughs off in 1-2 weeks. A preferred
embodiment of the instant invention for the treatment of cutaneous
anthrax is to administer a serine protease inhibitor (natural or
synthetic) in a topical/cream preparation. Parenteral serine
protease inhibitor therapy can also be co-administered in the event
that systemic symptoms emerge, or such parenteral therapy can be
administered prophylactically for anthrax that appears clinically
to be localized to the skin.
[0114] iii) Gastrointestinal anthrax appears after ingestion of
anthrax spores. After 2-5 days, one develops nausea/vomiting/fever,
and abdominal pain. Bloody diarrhea rapidly ensues, and an "acute
abdomen" manifests. The pathology within the abdomen includes
mucosal ulcerations. Also, hemorrhagic mesenteric lymphadenitis
develops, and this is again consistent with selective activation of
the anthrax toxin in serine protease-inhibitor deficient
microenvironments. This disease carries a mortality rate of
50%.
Isolated Proteins for Use in the Compositions and Methods of the
Invention
[0115] One aspect of the invention pertains to isolated proteins,
and biologically active portions thereof, as well as polypeptide
fragments suitable for use as immunogens to raise antibodies
directed against a polypeptide of the invention. In one embodiment,
the native polypeptide can be isolated from cells or tissue sources
by an appropriate purification scheme using standard protein
purification techniques. In another embodiment, polypeptides of the
invention are produced by recombinant DNA techniques. Alternative
to recombinant expression, a polypeptide of the invention can be
synthesized chemically using standard peptide synthesis
techniques.
[0116] Recombinant unmodified and mutant variants of
.alpha..sub.1-antitrypsin produced by genetic engineering methods
are also known (U.S. Pat. No. 4,711,848). The nucleotide sequence
of human .alpha.1-antitrypsin and other human .alpha.1-antitrypsin
variants has been disclosed in international published application
No. WO 86/00,337, the entire contents of which are incorporated
herein by reference. This nucleotide sequence may be used as
starting material to generate all of the AAT amino acid variants
and amino acid fragments depicted herein, using recombinant DNA
techniques and methods known to those of skill in the art.
[0117] An "isolated" or "purified" protein or biologically active
portion thereof is substantially free of cellular material or other
contaminating proteins from the cell or tissue source from which
the protein is derived, or substantially free of chemical
precursors or other chemicals when chemically synthesized. The
language "substantially free of cellular material" includes
preparations of protein in which the protein is separated from
cellular components of the cells from which it is isolated or
recombinantly produced. Thus, protein that is substantially free of
cellular material includes preparations of protein having less than
about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein
(also referred to herein as a "contaminating protein"). When the
protein or biologically active portion thereof is recombinantly
produced, it is also preferably substantially free of culture
medium, i.e., culture medium represents less than about 20%, 10%,
or 5% of the volume of the protein preparation. When the protein is
produced by chemical synthesis, it is preferably substantially free
of chemical precursors or other chemicals, i.e., it is separated
from chemical precursors or other chemicals which are involved in
the synthesis of the protein. Accordingly such preparations of the
protein have less than about 30%, 20%, 10%, 5% (by dry weight) of
chemical precursors or compounds other than the polypeptide of
interest.
[0118] Biologically active portions of a polypeptide of the
invention include polypeptides comprising amino acid sequences
sufficiently identical to or derived from the amino acid sequence
of the protein (e.g., the amino acid sequence shown in any of SEQ
ID NOS: 1, 2, 3, 4, 5, 6, 7; 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, which include fewer amino acids
than the full length protein, and exhibit at least one activity of
the corresponding full-length protein. Typically, biologically
active portions comprise a domain or motif with at least one
activity of the corresponding protein. A biologically active
portion of a protein of the invention can be a polypeptide which
is, for example, 10, 25, 50, 100 or more amino acids in length.
Moreover, other biologically active portions, in which other
regions of the protein are deleted, can be prepared by recombinant
techniques and evaluated for one or more of the functional
activities of the native form of a polypeptide of the
invention.
[0119] Preferred polypeptides have the amino acid sequence of SEQ
ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, and 60. Other useful proteins are
substantially identical (e.g., at least about 45%, preferably 55%,
65%, 75%, 85%, 95%, or 99%) to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, and 60, and retain the functional activity of the protein
of the corresponding naturally-occurring protein yet differ in
amino acid sequence due to natural allelic variation or
mutagenesis.
[0120] To determine the percent identity of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes (e.g., gaps can be introduced in the
sequence of a first amino acid or nucleic acid sequence for optimal
alignment with a second amino or nucleic acid sequence). The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position. The percent
identity between the two sequences is a function of the number of
identical positions shared by the sequences (i.e., % identity=# of
identical positions/total # of positions (e.g., overlapping
positions).times.100). In one embodiment, the two sequences are the
same length.
[0121] The determination of percent identity between two sequences
can be accomplished using a mathematical algorithm. A preferred,
non-limiting example of a mathematical algorithm utilized for the
comparison of two sequences is the algorithm of Karlin and Altschul
(1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in
Karlin and Altschul (1993) Proc. Natl. Acad. Sci. 90:5873-5877.
Such an algorithm is incorporated into the NBLAST and XBLAST
programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410.
BLAST nucleotide searches can be performed with the NBLAST program,
score=100, wordlength=12 to obtain nucleotide sequences homologous
to a nucleic acid molecules of the invention. BLAST protein
searches can be performed with the XBLAST program, score=50,
wordlength=3 to obtain amino acid sequences homologous to a protein
molecules of the invention. To obtain gapped alignments for
comparison purposes, Gapped BLAST can be utilized as described in
Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.
Alternatively, PSI-Blast can be used to perform an iterated search
which detects distant relationships between molecules (Id.). When
utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default
parameters of the respective programs (e.g., XBLAST and NBLAST) can
be used. See http://www.ncbi.nlm.nih.gov.
[0122] Another preferred, non-limiting example of a mathematical
algorithm utilized for the comparison of sequences is the algorithm
of Myers and Miller, CABIOS (1989). Such an algorithm is
incorporated into the ALIGN program (version 2.0) which is part of
the CGC sequence alignment software package. When utilizing the
ALIGN program for comparing amino acid sequences, a PAM120 weight
residue table, a gap length penalty of 12, and a gap penalty of 4
can be used. Additional algorithms for sequence analysis are known
in the art and include ADVANCE and ADAM as described in Torellis
and Robotti (1994) Comput. Appl. Biosci., 10:3-5; and FASTA
described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci.
85:2444-8. Within FASTA, ktup is a control option that sets the
sensitivity and speed of the search. If ktup=2, similar regions in
the two sequences being compared are found by looking at pairs of
aligned residues; if ktup=1, single aligned amino acids are
examined. ktup can be set to 2 or 1 for protein sequences, or from
1 to 6 for DNA sequences. The default if ktup is not specified is 2
for proteins and 6 for DNA. For a further description of FASTA
parameters, see
http://bioweb.pasteur.fr/d-ocs/Man/man/fasta.1.html#sect2, the
contents of which are incorporated herein by reference.
[0123] The percent identity between two sequences can be determined
using techniques similar to those described above, with or without
allowing gaps. In calculating percent identity, only exact matches
are counted.
[0124] The present invention also pertains to variants of the
polypeptides of the invention. Such variants have an altered amino
acid sequence which can function as either agonists (mimetics) or
as antagonists. Variants can be generated by mutagenesis, e.g.,
discrete point mutation or truncation. An agonist can retain
substantially the same, or a subset, of the biological activities
of the naturally occurring form of the protein. An antagonist of a
protein can inhibit one or more of the activities of the naturally
occurring form of the protein by, for example, competitively
binding to a downstream or upstream member of a cellular signaling
cascade which includes the protein of interest. Thus, specific
biological effects can be elicited by treatment with a variant of
limited function. Treatment of a subject with a variant having a
subset of the biological activities of the naturally occurring form
of the protein can have fewer side effects in a subject relative to
treatment with the naturally occurring form of the protein.
[0125] Variants of a protein of the invention which function as
either agonists (mimetics) or as antagonists can be identified by
screening combinatorial libraries of mutants, e.g., truncation
mutants, of the protein of the invention for agonist or antagonist
activity. In one embodiment, a variegated library of variants is
generated by combinatorial mutagenesis at the nucleic acid level
and is encoded by a variegated gene library. A variegated library
of variants can be produced by, for example, enzymatically ligating
a mixture of synthetic oligonucleotides into gene sequences such
that a degenerate set of potential protein sequences is expressible
as individual polypeptides, or alternatively, as a set of larger
fusion proteins (e.g., for phase display). There are a variety of
methods which can be used to produce libraries of potential
variants of the polypeptides of the invention from a degenerate
oligonucleotide sequence. Methods for synthesizing degenerate
oligonucleotides are known in the art (see, e.g., Narang (1983)
Tetrahedron 39:3; Itakura et al. (1984) Annu Rev. Biochem. 53:323;
Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic
Acid Res. 11:477).
[0126] In addition, libraries of fragments of the coding sequence
of a polypeptide of the invention can be used to generate a
variegated population of polypeptides for screening and subsequent
selection of variants. For example, a library of coding sequence
fragments can be generated by treating a double stranded PCR
fragment of the coding sequence of interest with a nuclease under
conditions wherein nicking occurs only about once per molecule,
denaturing the double stranded DNA, renaturing the DNA to form
double stranded DNA which can include sense/antisense pairs from
different nicked products, removing single stranded portions from
reformed duplexes by treatment with S1 nuclease, and ligating the
resulting fragment library into an expression vector. By this
method, an expression library can be derived which encodes
N-terminal and internal fragments of various sizes of the protein
of interest.
[0127] Several techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. The most widely used techniques, which
are amenable to high through-put analysis, for screening large gene
libraries typically include cloning the gene library into
replicable expression vectors, transforming appropriate cells with
the resulting library of vectors, and expressing the combinatorial
genes under conditions in which detection of a desired activity
facilitates isolation of the vector encoding the gene whose product
was detected. Recursive ensemble mutagenesis (REM), a technique
which enhances the frequency of functional mutants in the
libraries, can be used in combination with the screening assays to
identify variants of a protein of the invention (Arkin and Yourvan
(1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al.
(1993) Protein Engineering 6(3):327-331).
[0128] An isolated polypeptide of the invention, or a fragment
thereof, can be used as an immunogen to generate antibodies using
standard techniques for polyclonal and monoclonal antibody
preparation. The full-length polypeptide or protein can be used or,
alternatively, the invention provides antigenic peptide fragments
for use as immunogens. The antigenic peptide of a protein of the
invention comprises at least 8 (preferably 10, 15, 20, or 30) amino
acid residues of the amino acid sequence of SEQ ID NOs: 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, and 60, and encompasses an epitope of the protein such
that an antibody raised against the peptide forms a specific immune
complex with the protein.
Fusion Proteins for Use in the Compositions and Methods of the
Invention
[0129] In each of the aforementioned aspects and embodiments of the
invention, fusion polypeptides are also specifically contemplated
herein.
[0130] In one embodiment, fusion polypeptides of the invention are
produced by recombinant DNA techniques. Alternative to recombinant
expression, a fusion polypeptide of the invention can be
synthesized chemically using standard peptide synthesis techniques.
The present invention also provides compositions that comprise a
fusion polypeptide of the invention and a pharmaceutically
acceptable carrier, excipient or diluent.
[0131] In each of the above-recited methods, the mammalian
.alpha.1-antitrypsin or inhibitor of serine protease activity
substance may be part of a fusion polypeptide, wherein said fusion
polypeptide comprises mammalian .alpha.1-antitrypsin or inhibitor
of serine protease activity substance and an amino acid sequence
heterologous to said mammalian .alpha.1-antitrypsin or inhibitor of
serine protease activity substance.
[0132] Among the particular fusion polypeptides of the invention
are, for example, fusion polypeptides that comprise the amino acid
sequence of the .alpha.1-antitrypsin depicted below in SEQ ID
NO:63.
TABLE-US-00002 1 01 01 01 01 0 MPSSVSWGIL LAGLCCLVPV SLAEDPQGDA
AQKTDTSHHD QDHPTFNKIT PNLAEFAFSL YRQLAHQSNS TNIFFSPVSI ATAFAMLSLG
TKADTHDEIL 100 EGLNFNLTEI PEAQJHEGFQ ELLRTLNQPD SQLQLTTGNG
LFLSEGLKLV DKFLEDVKKL YHSEAFTVNF GDHEEAKKQI NDYVEKGTQG KIVDLVKELD
200 RDTVFALVNY IFFKGKWERP FEVKDTEDED FHVDQVTTVK VPMMKRLGMF
NIQHCKKLSS WVLLMKYLGN ATALFFLPDE GKLQHLENEL THDIITKYLE 300
NEDRRSASLH LPKLSITGTY DLKSVLGQLG ITKVFSNGAD LSGVTEEAPL KLSKAVHKAV
LTIDEKGTEA AGAMFLEAIP MSIPPEVKFN KPFVFLMIEQ 400 NTKSPLFMGK VVNPTQK
417
[0133] The fusion polypeptides of the invention can be such that
the heterologous amino acid sequence comprises a human
immunoglobulin constant region, such as a human IgG1 constant
region, including a modified human IgG1 constant region wherein the
IgG1 constant region does not bind Fc receptor and/or does not
initiate antibody-dependent cellular cytotoxicity (ADCC)
reactions.
[0134] In particular, in one embodiment the fusion protein
comprises a heterologous sequence that is a sequence derived from a
member of the immunoglobulin protein family, for example, comprise
an immunoglobulin constant region, e.g., a human immunoglobulin
constant region such as a human IgG1 constant region. The fusion
protein can, for example, comprise a portion of a mammalian
.alpha.1-antitrypsin or inhibitor of serine protease activity
polypeptide fused with the amino-terminus or the carboxyl-terminus
of an immunoglobulin constant region, as disclosed, e.g., in U.S.
Pat. No. 5,714,147, U.S. Pat. No. 5,116,964, U.S. Pat. No.
5,514,582, and U.S. Pat. No. 5,455,165. In those embodiments in
which all or part of a polypeptide of the invention is fused with
sequences derived from a member of the immunoglobulin protein
family, the FcR region of the immunoglobulin may be either
wild-type or mutated. In certain embodiments, it is desirable to
utilize an immunoglobulin fusion protein that does not interact
with a Fc receptor and does not initiate ADCC reactions. In such
instances, the immunoglobulin heterologous sequence of the fusion
protein can be mutated to inhibit such reactions. See, e.g., U.S.
Pat. No. 5,985,279 and WO 98/06248.
[0135] The heterologous amino acid sequence of the fusion
polypeptides utilized as part of the present invention can also
comprise an amino acid sequence useful for identifying, tracking or
purifying the fusion polypeptide, e.g., can comprise a FLAG or a
His tag sequence. The fusion polypeptide can further comprise an
amino acid sequence containing a proteolytic cleavage site which
can, for example, be useful for removing the heterologous amino
acid sequence from the .alpha.1-antitrypsin or inhibitor of serine
protease derivative or mimic sequence of the fusion
polypeptide.
[0136] In particular, the heterologous amino acid sequence of the
fusion polypeptides of the present invention can also comprise an
amino acid sequence useful for identifying, tracking or purifying
the fusion polypeptide, e.g., can comprise a FLAG (see, e.g., Hoop,
T. P. et al., Bio/Technology 6, 1204-1210 (1988); Prickett, K. S.
et al., BioTechniques 7, 580-589 (1989)) or a His tag (Van Reeth,
T. et al., BioTechniques 25, 898-904 (1998)) sequence. The fusion
polypeptide can further comprise an amino acid sequence containing
a proteolytic cleavage site which can, for example, be useful for
removing the heterologous amino acid sequence from the mammalian
.alpha.1-antitrypsin or inhibitor of serine protease activity
polypeptide sequence of the fusion polypeptide.
[0137] In yet another embodiment, the mammalian
.alpha.1-antitrypsin or inhibitor of serine protease-like activity
polypeptide fusion protein comprises a GST fusion protein in which
the mammalian .alpha.1-antitrypsin or inhibitor of serine protease
activity polypeptide of the invention is fused to the C-terminus of
GST sequences. Such a fusion protein can facilitate the
purification of a recombinant polypeptide of the invention. In
those embodiments in which a GST, FLAG or HisTag fusion constructs
is employed in the construction of the mammalian
.alpha.1-antitrypsin or inhibitor of serine protease activity
polypeptide fusion proteins, proteolytic cleavage sites may be
optionally introduced at the junction of the fusion moiety and the
mammalian .alpha.1-antitrypsin or inhibitor of serine protease
activity polypeptide to enable separation of the mammalian
.alpha.1-antitrypsin or inhibitor of serine protease activity
polypeptide from the fusion moiety subsequent to purification of
the mammalian .alpha.1-antitrypsin or inhibitor of serine protease
activity polypeptide. Such enzymes, and their cognate recognition
sequences, include, for example, without limitation, Factor Xa,
thrombin and enterokinase. Typical fusion expression vectors
include pGEX (Pharmacia Biotech Inc.; Smith and Johnson (1988) Gene
67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5
(Pharmacia, Piscataway, N.J.) which may be used to fuse glutathione
S-transferase (GST), maltose E binding protein, or protein A,
respectively, to the target mammalian .alpha.1-antitrypsin or
inhibitor of serine protease activity polypeptide protein.
[0138] Expression vectors can routinely be designed for expression
of a fusion polypeptide of the invention in prokaryotic (e.g., E.
coli) or eukaryotic cells (e.g., insect cells (using baculovirus
expression vectors), yeast cells or mammalian cells). Suitable host
cells are discussed further in Goeddel, supra. Alternatively, the
recombinant expression vector can be transcribed and translated in
vitro, for example using T7 promoter regulatory sequences and T7
polymerase.
[0139] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, in fusion expression vectors, a
proteolytic cleavage site is introduced at the junction of the
fusion moiety and the recombinant protein to enable separation of
the recombinant protein from the fusion moiety subsequent to
purification of the fusion protein. Such enzymes, and their cognate
recognition sequences, include Factor Xa, thrombin and
enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67:31-40),
pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,
Piscataway, N.J.) which fuse glutathione S-transferase (GST),
maltose E binding protein, or protein A, respectively, to the
target recombinant protein.
[0140] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET
lid (Studier et al., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89).
Target gene expression from the pTrc vector relies on host RNA
polymerase transcription from a hybrid trp-lac fusion promoter.
Target gene expression from the pET lid vector relies on
transcription from a T7 gn10-lac fusion promoter mediated by a
coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is
supplied by host strains BL21(DE3) or HMS174(DE3) from a resident
prophage harboring a T7 gn1 gene under the transcriptional control
of the lacUV 5 promoter.
[0141] One strategy to maximize recombinant protein expression in
E. coli is to express the protein in a host bacterium with an
impaired capacity to proteolytically cleave the recombinant protein
(Gottesman, Gene Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, Calif. (1990) 119-128). Another strategy
is to alter the nucleic acid sequence of the nucleic acid to be
inserted into an expression vector so that the individual codons
for each amino acid are those preferentially utilized in E. coli
(Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Such
alteration of nucleic acid sequences of the invention can be
carried out by standard DNA synthesis techniques.
[0142] In another embodiment, the expression vector is a yeast
expression vector. Examples of vectors for expression in yeast S.
cerivisae include pYepSec1 (Baldari et al. (1987) EMBO J.
6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943),
pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen
Corporation, San Diego, Calif.), and pPicZ (Invitrogen Corp, San
Diego, Calif.).
[0143] Alternatively, the expression vector is a baculovirus
expression vector. Baculovirus vectors available for expression of
proteins in cultured insect cells (e.g., Sf 9 cells) include the
pAc series (Smith et al. (1983) Mol. Cell. Biol. 3:2156-2165) and
the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
[0144] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression
vector. Examples of mammalian expression vectors include pCDM8
(Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO
J. 6:187-195). When used in mammalian cells, the expression
vector's control functions are often provided by viral regulatory
elements. For example, commonly used promoters are derived from
polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For
other suitable expression systems for both prokaryotic and
eukaryotic cells see chapters 16 and 17 of Sambrook et al.,
supra.
[0145] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al. (1987) Genes
Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton
(1988) Adv. Immunol. 43:235-275), in particular promoters of T cell
receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and
immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and
Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g.,
the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl.
Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund
et al. (1985) Science 230:912-916), and mammary gland-specific
promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and
European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, for
example the murine hox promoters (Kessel and Gruss (1990) Science
249:374-379) and the alpha-fetoprotein promoter (Campes and
Tilghman (1989) Genes Dev. 3:537-546).
[0146] A host cell can be any prokaryotic (e.g., E. coli) or
eukaryotic cell (e.g., insect cells, yeast or mammalian cells).
[0147] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid into a host cell, including
calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (supra), and other
laboratory manuals.
Combination Therapies for Treating Mycobacterial Diseases and
Anthrax Using the Methods of the Invention
[0148] In each of the aforementioned aspects and embodiments of the
invention, combination therapies other than those enumerated above
are also specifically contemplated herein. In particular, the
compositions of the present invention may be administered with one
or more macrolide or non-macrolide antibiotics, anti-bacterial
agents, anti-fungicides, anti-viral agents, and anti-parasitic
agents, anti-inflammatory or immunomodulatory drugs or agents.
[0149] Examples of macrolide antibiotics that may be used in
combination with the composition of the present invention include,
inter alia, the following synthetic, semi-synthetic or naturally
occurring microlidic antibiotic compounds: methymycin,
neomethymycin, YC-17, litorin, erythromycin A to F, oleandomycin,
roxithromycin, dirithromycin, flurithromycin, clarithromycin,
davercin, azithromycin, josamycin, kitasamycin, spiramycin,
midecamycin, rokitamycin, miokamycin, lankacidin, and the
derivatives of these compounds. Thus, erythromycin and compounds
derived from erythromycin belong to the general class of
antibiotics known as "macrolides." Examples of preferred
erythromycin and erythromycin-like compounds include: erythromycin,
clarithromycin, azithromycin, and troleandomycin.
[0150] Additional antibiotics, other than the macrolidic
antibiotics described above, which are suitable for use in the
methods of the present invention include, for example, any molecule
that tends to prevent, inhibit or destroy life and as such, and as
used herein, includes anti-bacterial agents, anti-fungicides,
anti-viral agents, and anti-parasitic agents. These agents may be
isolated from an organism that produces the agent or procured from
a commercial source (e.g., pharmaceutical company, such as Eli
Lilly, Indianapolis, Ind.; Sigma, St. Louis, Mo.).
[0151] For example, the anti-TB antibiotic isoniazid (isonicotinic
acid hydrazide) is frequently effective, but isoniazid often causes
severe, sometimes fatal, hepatitis. The risk of hepatitis increases
with the patient's age. Additionally, isoniazid causes peripheral
neuropathy in some recipients in a dose-related fashion. Rifampin,
another antibiotic used to treat TB, must be used in conjunction
with another drug such as isoniazid. This requirement for
combination therapy with rifampin applies to the initial treatment
as well as the retreatment of pulmonary TB.
[0152] Usually, isoniazid, rifampin, ethambutol and ethionamide are
given orally. Streptomycin is typically given intramuscularly.
Amikacin is given intramuscularly or intravenously. Clofazimine,
which is also used to treat leprosy, is given orally.
[0153] Amikacin is a semisynthetic aminoglycoside antibiotic
derived from Kanamycin A. For its preparation see U.S. Pat. No.
3,781,268. For a review see Kerridge, Pharmacological and
Biochemical Properties of Drug Substances 1:125-153, M. E.
Goldberg, ed. (1977). Amikacin is usually administered
intramuscularly or intravenously. For additional information
including clinical pharmacology, indications, side effects and
dosages, see the Physicians Desk Reference, 42 ed. (1988) at pages
744-746 (hereinafter, PDR).
[0154] Clofazimine is an antibacterial agent also known as
LAMPRENE.RTM. For its preparation, see Barry, et al., Nature
179:1013 (1957). For a review see Karat, et al., Brit. Med. J.
3:175 (1971). Clofazimine is generally given orally. For additional
information including clinical pharmacology, precautions and
dosages, see the PDR at page 982.
[0155] Ethionamide is an antibacterial agent also known as
AMIDAZINE.RTM. and TRECATOR.RTM. See British Patent No. 800,250.
This drug is typically given orally. For further information
including precautions and dosages, see the PDR at page 2310.
[0156] Ciprofloxacin is a broad spectrum synthetic antibacterial
agent for oral usage. It is also known as CIPRO.RTM. It is
typically given in total daily dosages of 500 to 1,000 milligrams
which is usually given in 2 equal doses in 24 hours. For further
information see the PDR (1989) at pages 1441-1443. other member of
this fluoroquinolone class of antibiotics include ofloxacin,
levofloxacin, troveofloxacin, pefloxacin, gatifloxacin, and
moxifloxacin.
[0157] Other examples of anti-bacterial antibiotic agents include,
but are not limited to, penicillins, cephalosporins, carbacephems,
cephamycins, carbapenems, monobactams, aminoglycosides,
glycopeptides, quinolones, tetracyclines, macrolides,
oxazalidinones, and fluoroquinolones. Examples of antibiotic agents
include, but are not limited to, Penicillin G (CAS Registry No.:
61-33-6); Methicillin (CAS Registry No.: 61-32-5); Nafcillin (CAS
Registry No.: 147-52-4); Oxacillin (CAS Registry No.: 66-79-5);
Cloxacillin (CAS Registry No.: 61-72-3); Dicloxacillin (CAS
Registry No.: 3116-76-5); Ampicillin (CAS Registry No.: 69-53-4);
Amoxicillin (CAS Registry No.: 26787-78-0); Ticarcillin (CAS
Registry No.: 34787-01-4); Carbenicillin (CAS Registry No.:
4697-36-3); Mezlocillin (CAS Registry No.: 51481-65-3); Azlocillin
(CAS Registry No.: 37091-66-0); Piperacillin (CAS Registry No.:
61477-96-1); Imipenem (CAS Registry No.: 74431-23-5); Aztreonam
(CAS Registry No.: 78110-38-0); Cephalothin (CAS Registry No.:
153-61-7); Cefazolin (CAS Registry No.: 25953-19-9); Cefaclor (CAS
Registry No.: 70356-03-5); Cefamandole formate sodium (CAS Registry
No.: 42540-40-9); Cefoxitin (CAS Registry No.: 35607-66-0);
Cefuroxime (CAS Registry No.: 55268-75-2); Cefonicid (CAS Registry
No.: 61270-58-4); Cefinetazole (CAS Registry No.: 56796-20-4);
Cefotetan (CAS Registry No.: 69712-56-7); Cefprozil (CAS Registry
No.: 92665-29-7); Loracarbef (CAS Registry No.: 121961-22-6);
Cefetamet (CAS Registry No.: 65052-63-3); Cefoperazone (CAS
Registry No.: 62893-19-0); Cefotaxime (CAS Registry No.:
63527-52-6); Ceftizoxime (CAS Registry No.: 68401-81-0);
Ceftriaxone (CAS Registry No.: 73384-59-5); Ceftazidime (CAS
Registry No.: 72558-82-8); Cefepime (CAS Registry No.: 88040-23-7);
Cefixime (CAS Registry No.: 79350-37-1); Cefpodoxime (CAS Registry
No.: 80210-62-4); Cefsulodin (CAS Registry No.: 62587-73-9);
Fleroxacin (CAS Registry No.: 79660-72-3); Nalidixic acid (CAS
Registry No.: 389-08-2); Norfloxacin (CAS Registry No.:
70458-96-7); Ciprofloxacin (CAS Registry No.: 85721-33-1);
Ofloxacin (CAS Registry No.: 82419-36-1); Enoxacin (CAS Registry
No.: 74011-58-8); Lomefloxacin (CAS Registry No.: 98079-51-7);
Cinoxacin (CAS Registry No.: 28657-80-9); Doxycycline (CAS Registry
No.: 564-25-0); Minocycline (CAS Registry No.: 10118-90-8);
Tetracycline (CAS Registry No.: 60-54-8); Amikacin (CAS Registry
No.: 37517-28-5); Gentamicin (CAS Registry No.: 1403-66-3);
Kanamycin (CAS Registry No.: 8063-07-8); Netilmicin (CAS Registry
No.: 56391-56-1); Tobramycin (CAS Registry No.: 32986-56-4);
Streptomycin (CAS Registry No.: 57-92-1); Azithromycin (CAS
Registry No.: 83905-01-5); Clarithromycin (CAS Registry No.:
81103-11-9); Erythromycin (CAS Registry No.: 114-07-8);
Erythromycin estolate (CAS Registry No.: 3521-62-8); Erythromycin
ethyl succinate (CAS Registry No.: 41342-53-4); Erythromycin
glucoheptonate (CAS Registry No.: 23067-13-2); Erythromycin
lactobionate (CAS Registry No.: 3847-29-8); Erythromycin stearate
(CAS Registry No.: 643-22-1); Vancomycin (CAS Registry No.:
1404-90-6); Teicoplanin (CAS Registry No.: 61036-64-4);
Chloramphenicol (CAS Registry No.: 56-75-7); Clindamycin (CAS
Registry No.: 18323-44-9); Trimethoprim (CAS Registry No.:
738-70-5); Sulfamethoxazole (CAS Registry No.: 723-46-6);
Nitrofurantoin (CAS Registry No.: 67-20-9); Rifampin (CAS Registry
No.: 13292-46-1); Mupirocin (CAS Registry No.: 12650-69-0);
Metronidazole (CAS Registry No.: 443-48-1); Cephalexin (CAS
Registry No.: 15686-71-2); Roxithromycin (CAS Registry No.:
80214-83-1); Co-amoxiclavuanate; combinations of Piperacillin and
Tazobactam; and their various salts, acids, bases, and other
derivatives.
[0158] Anti-fungal agents include, but are not limited to,
caspofungin, terbinafine hydrochloride, nystatin, amphotericin B,
griseofulvin, ketoconazole, miconazole nitrate, flucytosine,
fluconazole, itraconazole, clotrimazole, benzoic acid, salicylic
acid, and selenium sulfide.
[0159] Anti-viral agents include, but are not limited to,
valgancyclovir, amantadine hydrochloride, rimantadin, acyclovir,
famciclovir, foscamet, ganciclovir sodium, idoxuridine, ribavirin,
sorivudine, trifluridine, valacyclovir, vidarabin, didanosine,
stavudine, zalcitabine, zidovudine, interferon alpha, and
edoxudine.
[0160] Anti-parasitic agents include, but are not limited to,
pirethrins/piperonyl butoxide, permethrin, iodoquinol,
metronidazole, diethylcarbamazine citrate, piperazine, pyrantel
pamoate, mebendazole, thiabendazole, praziquantel, albendazole,
proguanil, quinidine gluconate injection, quinine sulfate,
chloroquine phosphate, mefloquine hydrochloride, primaquine
phosphate, atovaquone, co-trimoxazole
(sulfamethoxazole/trimethoprim), and pentamidine isethionate.
[0161] In another aspect, in the method of the present invention,
one may, for example, supplement the composition by administration
of a therapeutically effective amount of one or more an
anti-inflammatory or immunomodulatory drugs or agents. By
"immunomodulatory drugs or agents", it is meant, e.g., agents which
act on the immune system, directly or indirectly, e.g., by
stimulating or suppressing a cellular activity of a cell in the
immune system, e.g., T-cells, B-cells, macrophages, or antigen
presenting cells (APC), or by acting upon components outside the
immune system which, in turn, stimulate, suppress, or modulate the
immune system, e.g., hormones, receptor agonists or antagonists,
and neurotransmitters; immunomodulators can be, e.g.,
immunosuppressants or immunostimulants. By "anti-inflammatory
drugs", it is meant, e.g., agents which treat inflammatory
responses, i.e., a tissue reaction to injury, e.g., agents which
treat the immune, vascular, or lymphatic systems.
[0162] Anti-inflammatory or immunomodulatory drugs or agents
suitable for use in this invention include, but are not limited to,
interferon derivatives, e.g., betaseron, .beta.-interferon;
prostane derivatives, e.g., compounds disclosed in PCT/DE93/0013,
e.g., iloprost, cicaprost; glucocorticoid, e.g., cortisol,
prednisolone, methylprednisolone, dexamethasone; immunsuppressives,
e.g., cyclosporine A, FK-506, methoxsalene, thalidomide,
sulfasalazine, azathioprine, methotrexate; lipoxygenase inhibitors,
e.g., zileutone, MK-886, WY-50295, SC-45662, SC-41661A, BI-L-357;
leukotriene antagonists, e.g., compounds disclosed in DE 40091171
German patent application P 42 42 390.2; WO 9201675; SC-41930;
SC-50605; SC-51146; LY 255283 (D. K. Herron et al., FASEB J. 2:
Abstr. 4729, 1988); LY 223982 (D. M. Gapinski et al. J. Med. Chem.
33: 2798-2813, 1990); U-75302 and analogs, e.g., described by J.
Morris et al., Tetrahedron Lett. 29: 143-146, 1988, C. E. Burgos et
al., Tetrahedron Lett. 30: 5081-5084, 1989; B. M. Taylor et al.,
Prostaglandins 42: 211-224, 1991; compounds disclosed in U.S. Pat.
No. 5,019,573; ONO-LB-457 and analogs, e.g., described by K.
Kishikawa et al., Adv. Prostagl. Thombox. Leukotriene Res. 21:
407-410, 1990; M. Konno et al., Adv. Prostagl. Thrombox.
Leukotriene Res. 21: 411-414, 1990; WF-11605 and analogs, e.g.,
disclosed in U.S. Pat. No. 4,963,583; compounds disclosed in WO
9118601, WO 9118879; WO 9118880, WO 9118883, antiinflammatory
substances, e.g., NPC 16570, NPC 17923 described by L.
Noronha-Blab. et al., Gastroenterology 102 (Suppl.): A 672, 1992;
NPC 15669 and analogs described by R. M. Burch et al., Proc. Nat.
Acad. Sci. USA 88: 355-359, 1991; S. Pou et al., Biochem.
Pharmacol. 45: 2123-2127, 1993; peptide derivatives, e.g., ACTH and
analogs; soluble TNF-receptors; TNF-antibodies; soluble receptors
of interleukines, other cytokines, T-cell-proteins; antibodies
against receptors of interleukins, other cytokines, and
T-cell-proteins.
[0163] The therapeutic agents of the instant invention may be used
for the treatment of animal subjects or patients, and more
preferably, mammals, including humans, as well as mammals such as
non-human primates, dogs, cats, horses, cows, pigs, guinea pigs,
and rodents.
Modes of Administration
[0164] Modes of administration of the various therapeutic agents
used in the invention are exemplified below. However, the agents
can be delivered by any of a variety of routes including: by
injection (e.g., subcutaneous, intramuscular, intravenous,
intraarterial, intraperitoneal), by continuous intravenous
infusion, cutaenously, dermally, transdermally, orally (e.g.,
tablet, pill, liquid medicine), by implanted osmotic pumps (e.g.,
Alza Corp.), by suppository or aerosol spray.
[0165] The peptide-based serine protease inhibitors may be prepared
by any suitable synthesis method such as originally described by
Merrifield, J. Am. Chem. Soc., 85, p 2149 (1963). Synthetic
peptides which exhibit inhibitory activity toward serine proteases
and methods for preparing and using same are disclosed for example
in U.S. Pat. Nos. 4,829,052, 5,157,019 to Glover; U.S. Pat. No.
5,420,110 to Miller; U.S. Pat. No. 4,963,654 Katunuma as
incorporated herein by reference.
[0166] Those skilled in the art of biochemical synthesis will
recognize that for commercial-scale quantities of peptides, such
peptides are preferably prepared using recombinant DNA techniques,
synthetic techniques, or chemical derivatization of biologically or
chemically synthesized peptides.
[0167] The compounds of the present invention are used as
therapeutic agents in the treatment of a physiological (especially
pathological) condition caused in whole or part, by excessive
serine protease activity. The peptides may be administered as free
peptides or pharmaceutically acceptable salts thereof. The terms
used herein conform to those found in Budavari, Susan (Editor),
"The Merck Index" An Encyclopedia of Chemicals, Drugs, and
Biologicals; Merck & Co., Inc. The term "pharmaceutically
acceptable salt" refers to those acid addition salts or metal
complexes of the peptides which do not significantly or adversely
affect the therapeutic properties (e.g. efficacy, toxicity, etc.)
of the peptides. The peptides should be administered to individuals
as a pharmaceutical composition, which, in most cases, will
comprise the peptide and/or pharmaceutical salts thereof with a
pharmaceutically acceptable carrier. The term "pharmaceutically
acceptable carrier" refers to those solid and liquid carriers,
which do not significantly or adversely affect the therapeutic
properties of the peptides.
[0168] The pharmaceutical compositions containing peptides of the
present invention may be administered to individuals, particularly
humans, either intravenously, subcutaneously, intramuscularly,
intranasally, orally, topically, transdermally, parenterally,
gastrointestinally, transbronchially and transalveolarly. Topical
administration is accomplished via a topically applied cream, gel,
rinse, etc. containing therapeutically effective amounts of
inhibitors of serine proteases. Transdermal administration is
accomplished by application of a cream, rinse, gel, etc. capable of
allowing the inhibitors of serine proteases to penetrate the skin
and enter the blood stream. Parenteral routes of administration
include, but are not limited to, direct injection such as
intravenous, intramuscular, intraperitoneal or subcutaneous
injection. Gastrointestinal routes of administration include, but
are not limited to, ingestion and rectal. Transbronchial and
transalveolar routes of administration include, but are not limited
to, inhalation, either via the mouth or intranasally and direct
injection into an airway, such as through a tracheotomy,
tracheostomy, endotracheal tube, or metered dose or continuous
inhaler. In addition, osmotic pumps may be used for administration.
The necessary dosage will vary with the particular condition being
treated, method of administration and rate of clearance of the
molecule from the body.
[0169] Although the compounds described herein and/or their
derivatives may be administered as the pure chemicals, it is
preferable to present the active ingredient as a pharmaceutical
composition. The invention thus further provides the use of a
pharmaceutical composition comprising one or more compounds and/or
a pharmaceutically acceptable salt thereof, together with one or
more pharmaceutically acceptable carriers therefore and,
optionally, other therapeutic and/or prophylactic ingredients. The
carrier(s) must be acceptable in the sense of being compatible with
the other ingredients of the composition and not deleterious to the
recipient thereof.
[0170] Pharmaceutical compositions include those suitable for oral
or parenteral (including intramuscular, subcutaneous, cutaneous,
inhaled and intravenous) administration. The compositions may,
where appropriate, be conveniently presented in discrete unit
dosage forms and may be prepared by any of the methods well known
in the art of pharmacy. Such methods include the step of bringing
into association the active compound with liquid carriers, solid
matrices, semi-solid carriers, finely divided solid carriers or
combinations thereof, and then, if necessary, shaping the product
into the desired delivery system.
[0171] Pharmaceutical compositions suitable for oral administration
may be presented as discrete unit dosage forms such as hard or soft
gelatin capsules, cachets or tablets, each containing a
predetermined amount of the active ingredient; as a powder or as
granules; as a solution, a suspension or as an emulsion. The active
ingredient may also be presented as a bolus, electuary or paste.
Tablets and capsules for oral administration may contain
conventional excipients such as binding agents, fillers,
lubricants, disintegrants, or wetting agents. The tablets may be
coated according to methods well known in the art., e.g., with
enteric coatings.
[0172] Oral liquid preparations may be in the form of, for example,
aqueous or oily suspension, solutions, emulsions, syrups or
elixirs, or may be presented as a dry product for constitution with
water or another suitable vehicle before use. Such liquid
preparations may contain conventional additives such as suspending
agents, emulsifying agents, non-aqueous vehicles (which may include
edible oils), or preservative. The compounds may also be formulated
for parenteral administration (e.g., by injection, for example,
bolus injection or continuous infusion) and may be presented in
unit dose form in ampoules, pre-filled syringes, small bolus
infusion containers or in multi-dose containers with an added
preservative. The compositions may take such forms as suspensions,
solutions, or emulsions in oily or aqueous vehicles, and may
contain formulatory agents such as suspending, stabilizing and/or
dispersing agents. Alternatively, the active ingredient may be in
powder form, obtained by aseptic isolation of sterile solid or by
lyophilization from solution, for constitution with a suitable
vehicle, e.g., sterile, pyrogen-free water, before use.
[0173] For topical administration to the epidermis, the compounds
may be formulated as ointments, creams or lotions, or as the active
ingredient of a transdermal patch. Suitable transdermal delivery
systems are disclosed, for example, in Fisher et al. (U.S. Pat. No.
4,788,603) or Bawas et al. (U.S. Pat. Nos. 4,931,279, 4,668,504 and
4,713,224). Ointments and creams may, for example, be formulated
with an aqueous or oily base with the addition of suitable
thickening and/or gelling agents. Lotions may be formulated with an
aqueous or oily base and will in general also contain one or more
emulsifying agents, stabilizing agents, dispersing agents,
suspending agents, thickening agents, or coloring agents. The
active ingredient can also be delivered via iontophoresis, e.g., as
disclosed in U.S. Pat. No. 4,140,122, 4,383,529, or 4,051,842. At
least two types of release are possible in these systems. Release
by diffusion occurs when the matrix is non-porous. The
pharmaceutically effective compound dissolves in and diffuses
through the matrix itself. Release by microporous flow occurs when
the pharmaceutically effective compound is transported through a
liquid phase in the pores of the matrix.
[0174] Compositions suitable for topical administration in the
mouth include unit dosage forms such as lozenges comprising active
ingredient in a flavored base, usually sucrose and acacia or
tragacanth; pastilles comprising the active ingredient in an inert
base such as gelatin and glycerin or sucrose and acacia;
mucoadherent gels, and mouthwashes comprising the active ingredient
in a suitable liquid carrier.
[0175] When desired, the above-described compositions can be
adapted to provide sustained release of the active ingredient
employed, e.g., by combination thereof with certain hydrophilic
polymer matrices, e.g., comprising natural gels, synthetic polymer
gels or mixtures thereof.
[0176] The pharmaceutical compositions according to the invention
may also contain other adjuvants such as flavorings, coloring,
antimicrobial agents, or preservatives.
[0177] It will be further appreciated that the amount of the
compound, or an active salt or derivative thereof, required for use
in treatment will vary not only with the particular salt selected
but also with the route of administration, the nature of the
condition being treated and the age and condition of the patient
and will be selected, ultimately, at the discretion of the
attending physician.
[0178] A pharmaceutical composition of the invention contains an
appropriate pharmaceutically acceptable carrier as defined supra.
These compositions can take the form of solutions, suspensions,
tablets, pills, capsules, powders, sustained-release formulations
and the like. Suitable pharmaceutical carriers are described in
Remington's Pharmaceutical Sciences 1990, pp. 1519-1675, Gennaro,
A. R., ed., Mack Publishing Company, Easton, Pa. The serine
protease inhibitor molecules of the invention can be administered
in liposomes or polymers (see, Langer, R. Nature 1998, 392, 5).
Such compositions will contain an effective therapeutic amount of
the active compound together with a suitable amount of carrier so
as to provide the form for proper administration to the
subject.
[0179] In general, the compound is conveniently administered in
unit dosage form; for example, containing 5 to 2000 mg,
conveniently 10 to 1000 mg, most conveniently, 50 to 500 mg of
active ingredient per unit dosage form.
[0180] Desirable blood levels may be maintained by continuous
infusion to provide about 0.01-5.0 mg/kg/hr or by intermittent
infusions containing about 0.4-20 mg/kg of the active
ingredient(s). Buffers, preservatives, antioxidants and the like
can be incorporated as required.
[0181] The desired dose may conveniently be presented in a single
dose or as divided doses administered at appropriate intervals, for
example, as two, three, four or more sub-doses per day. The
sub-dose itself may be further divided, e.g., into a number of
discrete loosely spaced administrations, such as multiple
inhalations from an insufflator or by application of a plurality of
drops into the eye.
[0182] Actual dosage levels of active ingredients in the
pharmaceutical compositions of this invention may be varied so as
to obtain an amount of the active compound(s) that is effective to
achieve the desired therapeutic response for a particular patient,
compositions, and mode of administration. The selected dosage level
will depend upon the activity of the particular pharmaceutical
compound or analogue thereof of the present invention, the route of
administration, the severity of the condition being treated, and
the condition and prior medical history of the patient being
treated. However, it is within the skill of the art to start doses
of the pharmaceutical compound at levels lower than required to
achieve the desired therapeutic effect and to gradually increase
the dosage until the desired effect is achieved.
[0183] The pharmaceutical compositions of the present invention can
be used in both veterinary medicine and human therapy. The
magnitude of a prophylactic or therapeutic dose of the
pharmaceutical composition of the invention in the acute or chronic
management of pain associated with above-mentioned diseases or
indications will vary with the severity of the condition to be
treated and the route of administration. The dose, and perhaps the
dose frequency, will also vary according to the age, body weight,
and response of the individual patient. In general, the total daily
dose range of the pharmaceutical composition of this invention is
generally between about 1 to about 100 mg, preferably about 1 to
about 20 mg, and more preferably about 1 to about 10 mg of active
compound per kilogram of body weight per day are administered to a
mammalian patient. If desired, the effective daily dose may be
divided into multiple doses for purposes of administration, e.g.
two to four separate doses per day.
[0184] Alternatively, the total daily dose range of the active
ingredient of this invention should be sufficient to increase the
serum concentration of the protease inhibitor by 10-100
micromolar.
[0185] It is intended herein that by recitation of such specified
ranges, the ranges cited also include all those dose range amounts
between the recited range. For example, in the range about 1 and
100, it is intended to encompass 2 to 99, 3-98, etc, without
actually reciting each specific range. The actual preferred amounts
of the active ingredient will vary with each case, according to the
species of mammal, the nature and severity of the particular
affliction being treated, and the method of administration.
[0186] It is also understood that doses within those ranges, but
not explicitly stated, such as 30 mg, 50 mg, 75 mg, etc. are
encompassed by the stated ranges, as are amounts slightly outside
the stated range limits.
[0187] The actual preferred amounts of the active ingredient will
vary with each case, according to the species of mammal, the nature
and severity of the particular affliction being treated, and the
method of administration.
[0188] In general, the pharmaceutical compositions of the present
invention are periodically administered to an individual patient as
necessary to improve symptoms of the particular disease being
treated. The length of time during which the compositions are
administered and the total dosage will necessarily vary with each
case, according to the nature and severity of the particular
affliction being treated and the physical condition of the subject
or patient receiving such treatment.
[0189] It is further recommended that children, patients aged over
65 years, and those with impaired renal or hepatic function
initially receive low doses, and that they then be titrated based
on individual response(s) or blood level(s). It may be necessary to
use dosages outside these ranges in some cases, as will be apparent
to those of ordinary skill in the art. Further, it is noted that
the clinician or treating physician will know, with no more than
routine experimentation, how and when to interrupt, adjust, or
terminate therapy in conjunction with individual patient
response.
[0190] Useful dosages of the compounds of the present invention can
be determined by comparing their in vitro activity, and in vivo
activity in animal models. Methods for the extrapolation of
effective dosages in mice, and other animals, to humans are known
to the art; for example, see U.S. Pat. No. 4,938,949.
EXAMPLES
[0191] The following specific examples are provided to better
assist the reader in the various aspects of practicing the present
invention. As these specific examples are merely illustrative,
nothing in the following descriptions should be construed as
limiting the invention in any way. Such limitations are, or course,
defined solely by the accompanying claims.
Example 1
Effect of .alpha.1-Antitrypsin on Mycobacterium avium Complex (Mac)
Infection of Human Monocyte-Derived Macrophages
[0192] 1. TB or MAC organisms were suspended at a concentration of
one Mcfarland standard. One McFarland is defined as a degree of
turbidity of organisms suspended in liquid that matches that of a
standard aliquot. A sample turbidity that is equivalent to that of
the one McFarland standard represents about 10.sup.7 bacilli/ml.
The optimal duration of a test culture is approximately 10-12 days
of bacilli grown in Middlebrook 7H9 broth (=mycobacterium
medium).
[0193] 2. Infecting the cells. The cells infected were human
monocyte-derived macrophages (MDM). MDM were isolated from human
peripheral blood mononuclear cells (PBMC) that were obtained from
heparinized blood from healthy volunteers by centrifuging the
heparinized blood over a ficol-hypaque cushion. The isolated PBMC
were aliquoted into polystyrene tissue culture plates and the
monocytes are allowed to adhere.times.2 hrs (0.5.times.10.sup.6
PBMC were added to each well, of which approximately 10-20% are
monocytes). Experiments were performed in plates without or with
sterile round glass coverslips in the bottoms of the wells (see a.
below). Only the monocytic population within the PBMC will adhere
to the plates under these conditions. The wells were then washed
(to remove the non-adhering lymphocytes) and incubated in fresh
medium.times.10-12 days (medium=RPMI+10% fetal calf serum+100
units/ml of penicillin G), which allows maturation of the monocytes
into macrophages. The volume of medium in each well was 1.0 ml. The
medium was then removed from each well of MDM, and the wells were
replenished with either medium alone (control), with AAT, or with
ala-ala-pro-val-chloromethyl ketone (an AAT-like synthetic serine
protease inhibitor)(Bachem, Inc.), and the wells were incubated for
3.0 hr. Then, the MDM in each well were infected with MAC (strain
Mycobacterium avium 9141) or TB (strain H37RV) at a ratio of
mycobacterial bacilli/cell of 1.times.10.sup.6. After a 1.0 hr
incubation (to allow the mycobacteria to bind to the MDM surfaces),
the supernatants were removed and saved for cytokine assays. The
wells were then washed twice (with a 1:1 solution of RPMI and
saline),
Two independent assays were then used to quantify mycobacterial
infection of the human monocyte-derived macrophages:
[0194] a. Direct Observation and Counting of the Number of Infected
Cells in Each Well
[0195] For these experiments, the mycobacteria-infected MDM were
cultured in wells of a polystyrene tissue culture plate that had
sterile round glass cover slips inserted into the bottoms of the
wells. Since the MDMs were originally seeded onto these cover
slips, the MDMs adhered to the cover slip surfaces. After
incubation with MAC or TB, the wells were washed twice (as stated
above) and then fixed.times.1 hr using glutaraldehyde. The
mycobacteria were then stained using a mycobacterial stain
(Zeihl-Nielsson) without injuring the cells. The number of infected
cells was quantified optically and the data expressed as a percent
of the total number of MDM in each well.
[0196] b. Colony Counts
[0197] After the infected cells were washed twice (see above), the
cells in parallel wells that did not contain cover slips were lysed
using 1.0 ml of lysing buffer per well for 5.0 min (0.25% sDKF
lysis buffer).
[0198] After the infected MDM were lysed (see above), the lysate
fluid was diluted 1:1 with 1.0 ml of 7H9 medium. The mycobacterial
suspension was diluted serially 1:10 into 1% (vol/vol) 7H9 medium
and sterile water. The diluted mycobacterial suspensions were
vortexed and then 0.5 ml of the suspension from each aliquot was
plated onto mycobacteria medium (solid 7H9 medium). This
mycobacteria-containing fluid was then cultured. The plates were
incubated for 10-12 days for MAC and for 21-24 days for
Tuberculosis, and the number of mycobacterial colonies counted.
Results
Tuberculosis
Direct Observation Data
TABLE-US-00003 [0199] Control MDM ATT (5.0 mg/ml)- (no AAT).sup.a
exposed MDM Experiment 1 20% 4% Experiment 2 17% 6% .sup.apercent
of cells infected with m. tuberculosis.
Colony Count Data
[0200] In a separate experiment, we cultured the cell-associated TB
to independently confirm the inhibitory AAT effect. The TB counts
per ml were 1.6.times.10.sup.5 per ml in the control MDM cultures
and 0.57.times.10.sup.5 per ml in the AAT-exposed cultures, an
inhibitory effect of 64% due to the presence of AAT.
Mycobacterium avium Complex
[0201] We used the related mycobacterial organisms known as
mycobacterium avium complex (MAC). MAC is important because it is a
leading cause of infectious disease in AIDS patients. It is also a
difficult problem in normal people who contract this infection; it
is very difficult to treat, and sometimes impossible to treat with
current antimicrobial drugs. Using AAT or an AAT-like molecule may
represent a novel means of therapy in these infections.
Direct Observation Data
TABLE-US-00004 [0202] Control MDM ATT (5.0 mg/ml)- (no AAT).sup.a
exposed MDM Experiment 1 17% 10% .sup.apercent of cells infected
with m. tuberculosis.
Colony Count Data
[0203] FIG. 1 shows the results of 4 separate experiments that
demonstrate that AAT significantly blocks infection of MDM with MAC
with a mean effect of approximately 55% inhibition. These
experiments were conducted as described above. The AAT mimic refers
to ala-ala-pro-val-chloromethyl ketone (an AAT-like synthetic
serine protease inhibitor) (Supplier: Bachem). The AAT mimic
results confirm the AAT data using an independent species, and
provide proof that the concept of serine protease inhibition to
treat mycobacterial infections extends to small molecule inhibitors
that make attractive drug candidates.
[0204] In the same cultures depicted above, we measured the
concentration of the pro-inflammatory cytokine TNF.alpha.. As shown
in FIG. 2 and FIG. 3, AAT and the AAT mimic both significantly
inhibited the production of TNF.alpha. in the MDM cultures by up to
100%. The blockade of pro-inflammatory cytokine production may
represent an additional mechanism by which serine protease
inhibitors block infection with TB and with MAC.
Example 2
Clinical Study in MAC Infection
[0205] The data described above in vitro using MAC have been
supplemented with a clinical study. In this clinical investigation,
AAT phenotypes (alternative forms of the AAT protein) were assessed
in patients with documented lung infection with MAC and who had
lung disease. These patients were compared to a control group
consisting of patients with the lung disease bronchiectasis (in
order to show that the presence of lung disease alone did not
account for the presence of MAC infection).
TABLE-US-00005 MAC Infection Bronchiectasis N = 134 subjects
(lungs) (lung disease) P-value Sex Male 8.97% 23.21% Female 91.3%
76.79% Age (mean) 64.5 yrs 64.0 yrs ATT phenotype 0.006 (%
abnormal) YES 27.7% 5.3% NO 72.3% 94.7%
[0206] Note in this table that that for the control
(bronchiectasis) group, the proportion of patients with abnormal
AAT molecules is 5.3%. This is in marked contrast to the case in
the MAC.quadrature.infected group, where the proportion is 27.7%, a
5.2 fold increase. The MAC.quadrature.infected patients were 5.2
times as likely as the control group to harbor an abnormal form of
AAT. This establishes a clinical link between abnormal AAT
molecules and infection with MAC. Thus, the inhibitory role of
normal AAT that we discovered in vitro is borne out in
patients.
Example 3
Effect of .alpha.-1-Antitrypsin on Stimulated Interleukin-1 Beta
Production in Whole Human Blood
[0207] Design: Venipuncture was performed on 3 healthy volunteers
using a 21-gauge needle, and the venous blood was aspirated into a
heparinized tube. Blood was then aliquoted into 6 milliliter
polypropylene tubes and diluted 1:4 with sterile RPMI tissue
culture medium alone (Control), diluted 1:4 in medium containing
heat-killed Staphylococcus epidermidis at a final concentration of
1:1000 as a stimulus (Staph), or into tubes containing
Staphylococcus epidermidis and .alpha.-1-antitrypsin (AAT,
Aralast.RTM. from Baxter). All cultures were then
incubated.times.24 hrs at 37.degree. C./5% CO.sub.2). Following
incubation, the samples were centrifuged.times.1,500g, and the
supernatants collected. Supernatants were assayed for interleukin-1
beta concentration using a validated electrochemiluminescence
apparatus that quantifies cytokine proteins.
[0208] RESULTS: The data are presented as the mean.+-.SEM
interleukin-1 beta production, and the values are shown on the
vertical axis. As shown, AAT significantly inhibited
Staph-stimulated interleukin-1 beta production dose-dependently,
and the inhibition was observed at all concentrations tested (See
FIG. 5).
[0209] DISCUSSION: The inventors have shown herein for the first
time that AAT blocks IL-1 beta production as an example of
proinflammatory cytokine production. IL-1 beta is crucial for
development of the symptoms and/or manefestations of anthrax
disease. The results presented in this example supplement the
already supposed mechanism by which AAT may be used as a
therapeutic agent to cure anthrax by blocking the production of the
active toxin.
Example 4
[0210] In outpatient pneumonias, it is known that gram-positive
organisms predominate whereas in the intensive care unit (ICU),
gram-negative pneumonias are disproportionately incident.
[0211] The pathogenesis of pneumonia involves colonization followed
by micro-aspiration. Persons in the ICU become colonized with
gram-negative rods. Therefore, it is apparent to physicians that
only sick persons in the ICU become colonized with gram-negative
rods. Processed fibronectin is an important receptor for
gram-negative bacilli in vivo.
[0212] One means of treating patients with gram-negative pneumonias
would be to block gram-negative rod colonization. For example, in
health, unprocessed fibronectin is not a receptor for gram negative
bacteria. During illness, secretions become rich in serine
proteases. Serine proteases process (proteolize) fibronectin.
Processed fibronectin is a receptor for gram negative bacteria.
This results in colonization. The use of serine protease inhibitors
like .alpha.-1 antitrypsin or any of the functional derivatives
thereof as disclosed in this application can be used by one of
ordinary skill in the art to block gram-negative rod colonization
and therefore treat Gram-negative pneumonias. Thus, serine protease
inhibitors like AAT can be adminstered topically using topical
formulations including, for example, but not limited to, liquid,
cream, aerosol, etc., to block colonization of the epithelium by
Gram negative rods. Representative examples of publications
providing non-limiting examples of Gram negative bacilli that may
treated using the compositions of the present invention may be
found in Charlotte L. Barey-Morel et al. The Journal of Infectious
Diseases VI 155, No. 4 (1987); W. G. Johanson et al. Annals of
Internal Medicine 77: 701-706 (1972); W. G. Johanson et al. The New
England Journal of Medicine Vol 281 No. 21 (1969); James J. Rahal
et al. JAMA Vol. 214 No. 4 (1970), the entire texts of each of
which are incorporated by reference.
[0213] In a similar fashion serine protease inhibitors like AAT
could be administered topically using topical formulations
including, for example, but not limited to, liquid, cream, aerosol,
etc., to treat bacterial infections caused by Gram positive
organisms.
[0214] Likewise, in a similar fashion serine protease inhibitors
like AAT could be administered topically using topical formulations
including, for example, but not limited to, liquid, cream, aerosol,
etc., to treat bacterial infections caused by mycobacteria. For the
proposed mechanism of action for atypical mycobacteria, please
refer to Examples 1 and 2 supra.
[0215] Throughout this application various publications and patents
are referenced. The disclosures of these publications and patents
in their entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains.
[0216] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth, and as follows in the scope of the appended
claims.
Sequence CWU 1
1
6315PRTArtificialsynthetic peptide 1Phe Val Phe Leu Met1
525PRTArtificialsynthetic peptide 2Phe Val Phe Ala Met1
535PRTArtificialsynthetic peptide 3Phe Val Ala Leu Met1
545PRTArtificialsynthetic peptide 4Phe Val Phe Leu Ala1
555PRTArtificialsynthetic peptide 5Phe Leu Val Phe Ile1
565PRTArtificialsynthetic peptide 6Phe Leu Met Ile Ile1
575PRTArtificialsynthetic peptide 7Phe Leu Phe Val Leu1
585PRTArtificialsynthetic peptide 8Phe Leu Phe Val Val1
595PRTArtificialsynthetic peptide 9Phe Leu Phe Leu Ile1
5105PRTArtificialsynthetic peptide 10Phe Leu Phe Phe Ile1
5115PRTArtificialsynthetic peptide 11Phe Leu Met Phe Ile1
5125PRTArtificialsynthetic peptide 12Phe Met Leu Leu Ile1
5135PRTArtificialsynthetic peptide 13Phe Ile Ile Met Ile1
5145PRTArtificialsynthetic peptide 14Phe Leu Phe Cys Ile1
5155PRTArtificialsynthetic peptide 15Phe Leu Phe Ala Val1
5165PRTArtificialsynthetic peptide 16Phe Val Tyr Leu Ile1
5175PRTArtificialsynthetic peptide 17Phe Ala Phe Leu Met1
5185PRTArtificialsynthetic peptide 18Ala Val Phe Leu Met1
51910PRTHomo sapiens 19Met Pro Ser Ser Val Ser Trp Gly Ile Leu1 5
102010PRTHomo sapiens 20Leu Ala Gly Leu Cys Cys Leu Val Pro Val1 5
102110PRTHomo sapiens 21Ser Leu Ala Glu Asp Pro Gln Gly Asp Ala1 5
102210PRTHomo sapiens 22Ala Gln Lys Thr Asp Thr Ser His His Asp1 5
102310PRTHomo sapiens 23Gln Asp His Pro Thr Phe Asn Lys Ile Thr1 5
102410PRTHomo sapiens 24Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu1 5
102510PRTHomo sapiens 25Tyr Arg Gln Leu Ala His Gln Ser Asn Ser1 5
102610PRTHomo sapiens 26Thr Asn Ile Phe Phe Ser Pro Val Ser Ile1 5
102710PRTHomo sapiens 27Ala Thr Ala Phe Ala Met Leu Ser Leu Gly1 5
102810PRTHomo sapiens 28Thr Lys Ala Asp Thr His Asp Glu Ile Leu1 5
102910PRTHomo sapiens 29Glu Gly Leu Asn Phe Asn Leu Thr Glu Ile1 5
103010PRTHomo sapiens 30Pro Glu Ala Gln Ile His Glu Gly Phe Gln1 5
103110PRTHomo sapiens 31Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp1 5
103210PRTHomo sapiens 32Ser Gln Leu Gln Leu Thr Thr Gly Asn Gly1 5
103310PRTHomo sapiens 33Leu Phe Leu Ser Glu Gly Leu Lys Leu Val1 5
103410PRTHomo sapiens 34Asp Lys Phe Leu Glu Asp Val Lys Lys Leu1 5
103510PRTHomo sapiens 35Tyr His Ser Glu Ala Phe Thr Val Asn Phe1 5
103610PRTHomo sapiens 36Gly Asp His Glu Glu Ala Lys Lys Gln Ile1 5
103710PRTHomo sapiens 37Asn Asp Tyr Val Glu Lys Gly Thr Gln Gly1 5
103810PRTHomo sapiens 38Lys Ile Val Asp Leu Val Lys Glu Leu Asp1 5
103910PRTHomo sapiens 39Arg Asp Thr Val Phe Ala Leu Val Asn Tyr1 5
104010PRTHomo sapiens 40Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro1 5
104110PRTHomo sapiens 41Phe Glu Val Lys Asp Thr Glu Asp Glu Asp1 5
104210PRTHomo sapiens 42Phe His Val Asp Gln Val Thr Thr Val Lys1 5
104310PRTHomo sapiens 43Val Pro Met Met Lys Arg Leu Gly Met Phe1 5
104410PRTHomo sapiens 44Asn Ile Gln His Cys Lys Lys Leu Ser Ser1 5
104510PRTHomo sapiens 45Trp Val Leu Leu Met Lys Tyr Leu Gly Asn1 5
104610PRTHomo sapiens 46Ala Thr Ala Ile Phe Phe Leu Pro Asp Glu1 5
104710PRTHomo sapiens 47Gly Lys Leu Gln His Leu Glu Asn Glu Leu1 5
104810PRTHomo sapiens 48Thr His Asp Ile Ile Thr Lys Phe Leu Glu1 5
104910PRTHomo sapiens 49Asn Glu Asp Arg Arg Ser Ala Ser Leu His1 5
105010PRTHomo sapiens 50Leu Pro Lys Leu Ser Ile Thr Gly Thr Tyr1 5
105110PRTHomo sapiens 51Asp Leu Lys Ser Val Leu Gly Gln Leu Gly1 5
105210PRTHomo sapiens 52Ile Thr Lys Val Phe Ser Asn Gly Ala Asp1 5
105310PRTHomo sapiens 53Leu Ser Gly Val Thr Glu Glu Ala Pro Leu1 5
105410PRTHomo sapiens 54Lys Leu Ser Lys Ala Val His Lys Ala Val1 5
105510PRTHomo sapiens 55Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala1 5
105610PRTHomo sapiens 56Ala Gly Ala Met Phe Leu Glu Ala Ile Pro1 5
105710PRTHomo sapiens 57Met Ser Ile Pro Pro Glu Val Lys Phe Asn1 5
105810PRTHomo sapiens 58Lys Pro Phe Val Phe Leu Met Ile Glu Gln1 5
105910PRTHomo sapiens 59Asn Thr Lys Ser Pro Leu Phe Met Gly Lys1 5
10607PRTHomo sapiens 60Val Val Asn Pro Thr Gln Lys1 561394PRTHomo
sapiensMISC_FEATURE(355)..(358)native sequence 61Glu Asp Pro Gln
Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser His His1 5 10 15Asp Gln Asp
His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu 20 25 30Phe Ala
Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr 35 40 45Asn
Ile Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu 50 55
60Ser Leu Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu65
70 75 80Asn Phe Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly
Phe 85 90 95Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp Ser Gln Leu
Gln Leu 100 105 110Thr Thr Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu
Lys Leu Val Asp 115 120 125Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr
His Ser Glu Ala Phe Thr 130 135 140Val Asn Phe Gly Asp Thr Glu Glu
Ala Lys Lys Gln Ile Asn Asp Tyr145 150 155 160Val Glu Lys Gly Thr
Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu 165 170 175Asp Arg Asp
Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly 180 185 190Lys
Trp Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe 195 200
205His Val Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu
210 215 220Gly Met Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp
Val Leu225 230 235 240Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Ile
Phe Phe Leu Pro Asp 245 250 255Glu Gly Lys Leu Gln His Leu Glu Asn
Glu Leu Thr His Asp Ile Ile 260 265 270Thr Lys Phe Leu Glu Asn Glu
Asp Arg Arg Ser Ala Ser Leu His Leu 275 280 285Pro Lys Leu Ser Ile
Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly 290 295 300Gln Leu Gly
Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly305 310 315
320Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala
325 330 335Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala
Met Phe 340 345 350Leu Glu Ala Ile Pro Met Ser Ile Pro Pro Glu Val
Lys Phe Asn Lys 355 360 365Pro Phe Val Phe Leu Met Ile Glu Gln Asn
Thr Lys Ser Pro Leu Phe 370 375 380Met Gly Lys Val Val Asn Pro Thr
Gln Lys385 39062394PRTHomo sapiensMISC_FEATURE(355)..(358)novel
sequence 62Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser
His His1 5 10 15Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn
Leu Ala Glu 20 25 30Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln
Ser Asn Ser Thr 35 40 45Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Thr
Ala Phe Ala Met Leu 50 55 60Ser Leu Gly Thr Lys Ala Asp Thr His Asp
Glu Ile Leu Glu Gly Leu65 70 75 80Asn Phe Asn Leu Thr Glu Ile Pro
Glu Ala Gln Ile His Glu Gly Phe 85 90 95Gln Glu Leu Leu Arg Thr Leu
Asn Gln Pro Asp Ser Gln Leu Gln Leu 100 105 110Thr Thr Gly Asn Gly
Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp 115 120 125Lys Phe Leu
Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr 130 135 140Val
Asn Phe Gly Asp His Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr145 150
155 160Val Glu Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu
Leu 165 170 175Asp Arg Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe
Phe Lys Gly 180 185 190Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Thr
Glu Asp Glu Asp Phe 195 200 205His Val Asp Gln Val Thr Thr Val Lys
Val Pro Met Met Lys Arg Leu 210 215 220Gly Met Phe Asn Ile Gln His
Cys Lys Lys Leu Ser Ser Trp Val Leu225 230 235 240Leu Met Lys Tyr
Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp 245 250 255Glu Gly
Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile 260 265
270Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu
275 280 285Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val
Leu Gly 290 295 300Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gly Ala
Asp Leu Ser Gly305 310 315 320Val Thr Glu Glu Ala Pro Leu Lys Leu
Ser Lys Ala Val His Lys Ala 325 330 335Val Leu Thr Ile Asp Glu Lys
Gly Thr Glu Ala Ala Gly Ala Met Phe 340 345 350Leu Glu Arg Xaa Xaa
Arg Ser Ile Pro Pro Glu Val Lys Phe Asn Lys 355 360 365Pro Phe Val
Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe 370 375 380Met
Gly Lys Val Val Asn Pro Thr Gln Lys385 39063417PRTHomo sapiens
63Met Pro Ser Ser Val Ser Trp Gly Ile Leu Leu Ala Gly Leu Cys Cys1
5 10 15Leu Val Pro Val Ser Leu Ala Glu Asp Pro Gln Gly Asp Ala Ala
Gln 20 25 30Lys Thr Asp Thr Ser His His Asp Gln Asp His Pro Thr Phe
Asn Lys 35 40 45Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu Tyr
Arg Gln Leu 50 55 60Ala His Gln Ser Asn Ser Thr Asn Ile Phe Phe Ser
Pro Val Ser Ile65 70 75 80Ala Thr Ala Phe Ala Asn Leu Ser Leu Gly
Thr Lys Ala Asp Thr His 85 90 95Asp Glu Ile Leu Glu Gly Leu Asn Phe
Asn Leu Thr Glu Ile Pro Glu 100 105 110Ala Gln Ile His Glu Gly Phe
Gln Glu Leu Leu Arg Thr Leu Asn Gln 115 120 125Pro Asp Ser Gln Leu
Gln Leu Thr Thr Gly Asn Gly Leu Phe Leu Ser 130 135 140Glu Gly Leu
Lys Leu Val Asp Lys Phe Leu Glu Asp Val Lys Lys Leu145 150 155
160Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp His Glu Glu Ala
165 170 175Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr Gln Gly
Lys Ile 180 185 190Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr Val
Phe Ala Leu Val 195 200 205Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu
Arg Pro Phe Glu Val Lys 210 215 220Asp Thr Glu Asp Glu Asp Phe His
Val Asp Gln Val Thr Thr Val Lys225 230 235 240Val Pro Met Met Lys
Arg Leu Gly Met Phe Asn Ile Gln His Cys Lys 245 250 255Lys Leu Ser
Ser Trp Val Leu Leu Met Lys Tyr Leu Gly Asn Ala Thr 260 265 270Ala
Ile Phe Phe Leu Pro Asp Glu Gly Lys Leu Gln His Leu Glu Asn 275 280
285Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu Glu Asn Glu Asp Arg
290 295 300Arg Ser Ala Ser Leu His Leu Pro Lys Leu Ser Ile Thr Gly
Thr Tyr305 310 315 320Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile
Thr Lys Val Phe Ser 325 330 335Asn Gly Ala Asp Leu Ser Gly Val Thr
Glu Glu Ala Pro Leu Lys Leu 340 345 350Ser Lys Ala Val His Lys Ala
Val Leu Thr Ile Asp Glu Lys Gly Thr 355 360 365Glu Ala Ala Gly Ala
Met Phe Leu Glu Ala Ile Pro Met Ser Ile Pro 370 375 380Pro Glu Val
Lys Phe Asn Lys Pro Phe Val Phe Leu Met Ile Glu Gln385 390 395
400Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val Asn Pro Thr Gln
405 410 415Lys
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References