U.S. patent application number 10/658112 was filed with the patent office on 2005-06-02 for treatment of mycobacterial diseases by administration of bacterial/permeability-increasing protein products.
This patent application is currently assigned to XOMA CORPORATION. Invention is credited to Lambert, Lewis H. JR..
Application Number | 20050118112 10/658112 |
Document ID | / |
Family ID | 26706889 |
Filed Date | 2005-06-02 |
United States Patent
Application |
20050118112 |
Kind Code |
A1 |
Lambert, Lewis H. JR. |
June 2, 2005 |
Treatment of Mycobacterial diseases by administration of
bacterial/permeability-increasing protein products
Abstract
The present invention relates to methods for treating a subject
suffering from infection with Mycobacteria, such as M. leprae or M.
tuberculosis comprising administering to the subject a composition
comprising a bactericidal/permeability-inducing (BPI) protein
product alone or in combination with administration of an
anti-Mycobacterial antibiotic.
Inventors: |
Lambert, Lewis H. JR.;
(Fremont, CA) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN LLP
6300 SEARS TOWER
233 S. WACKER DRIVE
CHICAGO
IL
60606
US
|
Assignee: |
XOMA CORPORATION
Berkeley
CA
|
Family ID: |
26706889 |
Appl. No.: |
10/658112 |
Filed: |
September 8, 2003 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10658112 |
Sep 8, 2003 |
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09782642 |
Feb 13, 2001 |
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6620785 |
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09782642 |
Feb 13, 2001 |
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08626646 |
Apr 1, 1996 |
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6214789 |
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08626646 |
Apr 1, 1996 |
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08285803 |
Aug 4, 1994 |
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08285803 |
Aug 4, 1994 |
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08031145 |
Mar 12, 1993 |
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Current U.S.
Class: |
424/46 ; 514/2.2;
514/2.4 |
Current CPC
Class: |
Y10S 530/827 20130101;
A61K 38/1751 20130101; Y10S 514/924 20130101 |
Class at
Publication: |
424/046 ;
514/008 |
International
Class: |
A61K 038/17; A61L
009/04; A61K 009/14 |
Claims
1-9. (canceled)
10. A method of treating a subject suffering from the adverse
physiological effects of the presence of lipoarabinomannan in
circulation, the method comprising administering to the subject a
pharmaceutical composition comprising a BPI protein product
selected from the group consisting of amino-terminal fragments of
BPI holoprotein having a molecular weight of 21-25 kD by SDS-PAGE
and dimeric forms thereof, rBPI.sub.21, rBPI.sub.23, rBPI and
BPI-derived peptides, or fusion protein comprising said BPI protein
product, and a pharmaceutically acceptable diluent, adjuvant, or
carrier.
11. The method of claim 10 wherein the adverse physiological
effects comprise compromised immune response to microbes or tumor
cells due to lipoarabinomannan-induced inhibition of macrophage
activation by T-cell lymphokines.
12. The method of claim 10 wherein the adverse physiological
effects comprise increased production of a cytokine by the
subject.
13. The method of claim 10 wherein the composition is administered
orally.
14. The method of claim 10 wherein the composition is administered
intravenously.
15. The method of claim 10 wherein the composition is administered
as an aerosol.
16. The method of claim 10 wherein the BPI protein product is a
21-25 kD amino-terminal fragment of
Bactericidal/permeability-increasing protein.
17. The method of claim 10, wherein the composition further
comprises a surfactant.
18-23. (canceled)
Description
[0001] This is a continuation-in-part of co-pending U.S. patent
application Ser. No. 08/031,145, filed Mar. 12, 1993.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to methods of treating a
subject suffering from infection with Mycobacteria by
administration of Bactericidal/Permeability-Increasing Protein
(BPI) protein products. Mycobacterium is a non-motile, acid-fast,
aerobic, genus of bacteria known to cause grave human and animal
diseases, such as tuberculosis and leprosy. Infections caused by M.
avium are the most common form of disseminated bacterial disease in
AIDS patients. Orme, et al., Infect. and Immun., 61 (1): 338-342
(1993).
[0003] The administration of conventional antibiotics to treat
Mycobacterial infection is known in the art and has achieved
varying success depending on the susceptibility of the bacterial
strain, the efficacy and toxicity of the antibiotic(s) employed,
the duration of treatment, and numerous other factors.
Antimicrobials that have been employed alone or in combination to
treat Mycobacterial infections, including those caused by M.
tuberculosis include isoniazid, rifampin, ethambutol,
p-aminosalicylic acid, pyrazinamide, streptomycin, capreomycin,
cycloserine, ethionamide, kanamycin, amikacin, amithiozone,
rifabutin, clofazimine, arithromycin, clarithromycin, ciprofloxacin
and ofloxacin. McClatchy, Antimycobacterial Drugs: Mechanisms of
Action, Drug Resistance, Susceptibility Testing, and Assays of
Activity in Biological Fluids, pp. 134-197, In Antibiotics in
Laboratory Medicine, 3rd ed., V. Lorian, ed. The Williams &
Wilkins Co., Baltimore (1991). As many Mycobacterial strains are
drug resistant, serious obstacles exist for control and successful
treatment of tuberculosis and other Mycobactenal diseases. Id.
[0004] A variety of factors have made treatment of individuals
afflicted with Mycobactenal diseases problematic. First,
Mycobacteria possess a very hydrophobic cell wall that affords
protection against the host's immune system. As Mycobacterial
infections tend to be chronic, the pathologies of these organisms
are generally due to host response. Also, many Mycobacterial
strains are drug-resistant. These and other factors make the
development of novel, effective methods for treating Mycobacterial
diseases highly desirable.
[0005] Mycobacteria are readily distinguished from gram-negative
and gram-positive bacteria by acid fast staining due to significant
differences in cell wall structure. Gram-negative bacteria are
characterized by a cell wall composed of a thin layer of
peptidoglycan covered by an outer membrane of lipoprotein and
lipopolysaccharide (LPS), whereas gram-positive bacteria have a
cell wall with a thicker layer of peptidoglycan with attached
teichoic acids, but no LPS. The Mycobacterial cell wall is rich in
fatty acids, including a major constituent, lipoarabinomannan
(LAM), which is widely distributed within the cell wall of
Mycobacterium species. LAM has been purified from both M. leprae
and M. tuberculosis. Hunter et al., J. Biol. Chem., 261:
12345-12351 (1986). LAM is a serologically active mannose
containing phosphorylated lipopolysaccharide that may be membrane
associated.
[0006] The complex physiological effects of LAM appear to be
concentration, time, and source-dependent. For example, Chaterjee
et al., Infect. and Immun., 60 (3): 1249-1253 (1992), reported
that, in the first 24 hours following exposure, LAM from an
avirulent strain of tuberculosis was 100-fold more potent at
stimulating TNF secretion in mouse macrophages than LAM from a
virulent strain. LAM concentrations of 0.01-10 .mu.g/ml for the
avirulent strain and 0.01-100 .mu.g/ml for the virulent strain were
tested, and increased LAM concentration was associated with
increased TNF production with LAM from both species.
[0007] Macrophage-inhibitory effects of LAM have also been
described in the art. LAM purified from both M. leprae and M.
tuberculosis has been reported to be a potent in vitro inhibitor of
T-cell lymphokine activation of mouse macrophages. Sibley et al.,
Infection and Immunity, 56 (5): 1232-1236 (1988). Because the
principle efferent role of the macrophage in acquired resistance to
intracellular pathogens requires activation by T-cell lymphokines,
notably gamma-interferon (IFN-.gamma.), macrophages whose
activation-response is inhibited are severely compromised in their
capacity for both enhanced microbicidal and tumoricidal
activities.
[0008] In another study, Sibley et al., Clin. Exp. Immunol., 80
(1): 141-148 (1990), reported that pretreatment of mouse
macrophages with 50 to 100 ug/ml LAM blocked macrophage activation
by IFN-.gamma., but pretreatment with 10 .mu.g/ml LAM did not
affect macrophage activation. Thus, it is believed that low
concentrations of LAM stimulate cytokine production, at least
initially. However, higher concentrations of LAM (50-100 .mu.g/ml
or more) appear to block rather than promote macrophage function.
Thus, the production of either too much or too little cytokine at
different stages of Mycobacterial disease may contribute to
Mycobacterial pathogenesis. New methods for blocking the
above-characterized physiological effects of LAM molecules are a
highly desirable goal in the treatment of subjects that are or that
have been infected with Mycobacteria. For the same reasons, new
methods by which fluids containing LAM can be decontaminated prior
to administration into a subject are also desirable. Neutralization
of even small amounts of LAM is desirable, because small amounts of
LAM may have the physiological effect of stimulating cytokine
production.
[0009] Of interest to the background of the invention are the
disclosures of PCT/US88/00510, (WO 88/06038) published Aug. 25,
1988, indicating that certain poloxypropylene/polyoxyethylene
nonionic surface-active block copolymers can be used with or
without conventional antibiotics to treat infection with
Mycobacterium. This reference cites studies suggesting that the
effects of other nonionic surfactants on tuberculosis are most
likely due to modification of surface lipids of Mycobacteria, and
not to direct bactericidal effects on Mycobacteria. See e.g.
Cornforth et al., Nature, 168: 150-153 (1951).
[0010] Bactericidal/permeability-increasing protein (BPI) is a
protein isolated from the granules of mammalian polymorphonuclear
neutrophils (PMN), which are blood cells essential in the defense
against invading microorganisms. Human BPI protein has been
isolated from PMN's by acid extraction combined with either ion
exchange chromatography Elsbach, J. Biol. Chem., 254: 11000 (1979)
or E. coli affinity chromatography, Weiss, et al., Blood, 69: 652
(1987), and has potent bactericidal activity against a broad
spectrum of gram-negative bacteria. The molecular weight of human
BPI is approximately 55,000 Daltons (55 kD). The amino acid
sequence of the entire human BPI protein, as well as the DNA
encoding the protein, have been elucidated in FIG. 1 of Gray, et
al., J. Biol. Chem., 264: 9505 (1989), incorporated herein by
reference.
[0011] BPI has been shown to be a potent bactericidal agent active
against a broad range of gram-negative bacterial species. The
cytotoxic effect of BPI was originally established to be highly
specific to sensitive gram-negative species, with no toxicity being
noted for other non-acid fast, gram-positive bacteria or for
eukaryotic cells. The precise mechanism by which BPI kills bacteria
is as yet unknown, but it is known that BPI must first attach to
the surface of susceptible gram-negative bacteria. It is thought
that this initial binding of BPI to the bacteria involves
electrostatic interactions between the basic BPI protein and
negatively charged sites on lipopolysaccharides (LPS). LPS has been
referred to as endotoxin because of the potent inflammatory
response that it stimulates. LPS induces the release of mediators
by host inflammatory cells which may ultimately result in
irreversible endotoxic shock. BPI binds to Lipid A, the most toxic
and most biologically active component of LPS.
[0012] In susceptible bacteria, it is thought that BPI binding
disrupts LPS structure, leads to an activation of bacterial enzymes
that degrade phospholipids and peptidoglycans, alters the
permeability of the cell's outer membrane, and ultimately causes
cell death by an as yet unknown mechanism. BPI is also capable of
neutralizing the endotoxic properties of LPS to which it binds.
Because of its gram-negative bactericidal properties and its
ability to neutralize LPS, BPI can be utilized for the treatment of
mammals suffering from diseases caused by gram-negative bacteria,
such as bacteremia or sepsis.
[0013] An approximately 25 kD proteolytic fragment corresponding to
the amino-terminal portion of human BPI holoprotein possesses the
antibacterial efficacy of the naturally-derived 55 kD human
holoprotein. In contrast to the amino-terminal portion the
carboxy-terminal region of the isolated human BPI protein displays
only slightly detectable anti-bacterial activity. Ooi, et al., J.
Exp. Med., 174: 649 (1991). A BPI amino-terminal fragment,
expressed from a construct encoding approximately the first 199
amino acid residues of the human BPI holoprotein, has been produced
by recombinant means as a 23 kD protein referred to as
"rBPI.sub.23". Gazzano-Santoro et al., Infect. Immun. 60: 4754-4761
(1992).
[0014] While BPI protein products are effective for treatment of
conditions associated with gram-negative bacterial infection, there
continues to exist a need in the art for products and methods for
treatment of other bacterial infections such as infection with
Mycobacteria.
SUMMARY OF THE INVENTION
[0015] The present invention provides methods of treating a subject
suffering from infection with Mycobacteria by administration of a
composition comprising a BPI protein product. Therapeutic
compositions according to the invention may be administered orally,
systemically (such as by intravenous, intramuscular or other
injection), or as an aerosol. Mycobacterial disease states subject
to treatment according to the invention include tuberculosis, which
can be caused by infection with M. tuberculosis, leprosy, which can
be caused by infection with M. leprae, and diseases caused by M.
avium and other Mycobacteria species. According to preferred
methods, anti-Mycobacterial antibiotics such as previously
identified and/or surfactants may be administered in combination
with the BPI protein product to subjects suffering from infection
with Mycobacteria.
[0016] According to another aspect of the present invention,
compositions comprising a BPI protein product are administered to
neutralize LAM's physiological effects on a host. For example,
methods are provided for neutralizing the effect of low
concentrations of LAM capable of stimulating cytokine production in
a host. Methods are also provided for neutralizing the inhibitory
effect that higher concentrations of Mycobacterial LAM (i.e. 100
.mu.g/ml or more) have upon the interferon-mediated activation of
macrophages. Specifically, a BPI protein product may be
administered to an immunosuppressed subject failing to respond to
microbes or tumor cells due to LAM-induced insensitivity of
macrophages to activation by T-cell lymphokines.
[0017] According to a further aspect of the present invention, a
BPI protein product is employed in methods for decontaminating a
fluid containing LAM prior to administration of the fluid into a
subject. Such decontamination methods of the invention involve
contacting the fluid with the BPI protein product prior to
administration, under conditions such that LAM forms a complex with
the BPI protein product which can be removed from the fluid. Fluids
subject to decontamination by the methods of this invention
include, but are not limited to, blood, plasma, blood serum, bone
marrow, isotonic solutions, pharmaceutical agents, and cell culture
agents.
[0018] A further aspect of this invention relates to the use of a
composition comprising a BPI protein product for the manufacture of
a medicament for the therapeutic application of treating any of the
aforementioned conditions or infections from which a subject might
suffer.
[0019] Numerous additional aspects and advantages of the invention
will become apparent to those skilled in the art upon considering
the following detailed description of the invention, which
describes presently preferred embodiments thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 graphically depicts the results of an assay of BPI
protein product binding to E. coli J5 Lipid A and M. tuberculosis
and
[0021] FIG. 2 graphically represents the results of test to assess
the ability of a BPI protein product to inhibit mycobacterial
induced TNF production in whole blood.
DETAILED DESCRIPTION OF THE INVENTION
[0022] The present invention relates to the discovery that a
composition comprising a BPI protein product can be administered
for effective treatment of a subject suffering from infection with
Mycobacteria. In particular, the invention provides methods for
treatment of leprosy and tuberculosis, grave diseases caused by the
species M. leprae and M. tuberculosis, respectively. It is
contemplated that the methods described herein may be used to treat
infection with other Mycobacterial species, most notably M. avium
and M. intracullulare (collectively known as "MAC"), but also M.
marinum, M. fortuitum, M. chelonae, M. smegmatis, M. kansasii, M.
bovis, M. hominis, M. gordonae and other parthogenic or
opportunistic species. Beneficial effects of treatment with BPI
protein products are expected to result from binding of the
products to LAM and disruption of the bacterial cell wall
components (with or without direct killing of the bacteria) in
manner similar to that resulting from treatment of gram-negative
disease states.
[0023] As used herein, "BPI protein product" includes naturally and
recombinantly produced BPI protein; natural, synthetic, and
recombinant biologically active polypeptide fragments of BPI
protein; biologically active polypeptide variants of BPI protein or
fragments thereof, including hybrid fusion proteins and dimers; and
biologically active polypeptide analogs of BPI protein or fragments
or variants thereof, including cysteine-substituted analogs. The
BPI protein products administered according to this invention may
be generated and/or isolated by any means known in the art. U.S.
Pat. No. 5,198,541, the disclosure of which is hereby incorporated
by reference, discloses recombinant genes encoding and methods for
expression of BPI proteins including recombinant BPI holoprotein,
referred to herein as rBPI.sub.50 and recombinant fragments of BPI.
Co-owned, copending U.S. patent application Ser. No. 07/885,501 and
a continuation-in-part thereof, U.S. patent application Ser. No.
08/072,063 filed May 19, 1993 which are hereby incorporated by
reference, disclose novel methods for the purification of
recombinant BPI protein products expressed in and secreted from
genetically transformed mammalian host cells in culture and
discloses how one may produce large quantities of recombinant BPI
products suitable for incorporation into stable, homogeneous
pharmaceutical preparations.
[0024] Biologically active fragments of BPI (BPI fragments) include
biologically active molecules that have the same amino acid
sequence as a natural human BPI holoprotein, except that the
fragment molecule lacks amino-terminal amino acids, internal amino
acids, and/or carboxy-terminal amino acids of the holoprotein.
Nonlimiting examples of such fragments include an N-terminal
fragment of natural human BPI of approximately 25 kD, described in
Ooi et al., J. Exp. Med., 174: 649 (1991), and the recombinant
expression product of DNA encoding N-terminal amino acids from
residue 1 to about residue 200, including from about residue 1 to
about residue 193 or 199 of natural human BPI, described in
Gazzano-Santoro et al., Infect. Immun. 60: 4754-4761 (1992), and
referred to as rBPI.sub.23. In that publication, an expression
vector was used as a source of DNA encoding a recombinant
expression product (rBPI.sub.23) having the 31-residue signal
sequence and the first 199 amino acids of the N-terminus of the
mature human BPI, as set out in FIG. 1 of Gray et al., supra,
except that valine at position 151 is specified by GTG rather than
GTC and residue 185 is glutamic acid (specified by GAG) rather than
lysine (specified by AAG). Recombinant holoprotein (rBPI) has also
been produced having the sequence (SEQ. ID NOS: 1 and 2) set out in
FIG. 1 of Gray et al., supra, with the exceptions noted for
rBPI.sub.23 and with the exception that residue 417 is alanine
(specified by GCT) rather than valine (specified by GTT). Other
examples include dimeric BPI forms as described in co-owned and
co-pending U.S. patent application Ser. No. 08/212,132, filed Mar.
11, 1994, the disclosure of which is hereby incorporated by
reference.
[0025] Biologically active variants of BPI (BPI variants) include
but are not limited to recombinant hybrid fusion proteins,
comprising BPI holoprotein or a biologically active fragment
thereof and at least a portion of at least one other polypeptide,
and dimeric forms of BPI variants. Examples of such hybrid fusion
proteins and dimeric forms are described by Theofan et al. in
co-owned, copending U.S. patent application Ser. No. 07/885,911,
and a continuation-in-part application thereof U.S. patent
application Ser. No. 08/064,693 filed May 19, 1993 which are
incorporated herein by reference in their entirety and include
hybrid fusion proteins comprising, at the amino-terminal end, a BPI
protein or a biologically active fragment thereof and, at the
carboxy-terminal end, at least one constant domain of an
immunoglobulin heavy chain or allelic variant thereof.
[0026] Biologically active analogs of BPI (BPI analogs) include but
are not limited to BPI protein products wherein one or more amino
acid residue has been replaced by a different amino acid. For
example, co-owned, copending U.S. patent application Ser. No.
08/013,801 (Theofan et al., "Stable
Bactericidal/Permeability-Increasing Protein Products and
Pharmaceutical Compositions Containing the Same," filed Feb. 2,
1993), the disclosure of which is incorporated herein by reference,
discloses polypeptide analogs of BPI and BPI fragments wherein a
cysteine residue is replaced by a different amino acid. A preferred
BPI protein product described by this application is the expression
product of DNA encoding from amino acid 1 to approximately 193 or
199 of the N-terminal amino acids of BPI holoprotein, but wherein
the cysteine at residue number 132 is substituted with alanine and
is designated rBPI.sub.21.DELTA.cys or rBPI.sub.21.
[0027] Other BPI protein products useful according to the methods
of the invention are peptides derived from or based on BPI produced
by recombinant or synthetic means (BPI-derived peptides), such as
those described in co-owned and copending U.S. patent application
Ser. No. 08/209,762, filed Mar. 11, 1994, which is a
continuation-in-part of U.S. patent application Ser. No.
08/183,222, filed Jan. 14, 1994, which is a continuation-in-part of
U.S. patent application Ser. No. 08/093,202 filed Jul. 15, 1993),
which is a continuation-in-part of U.S. patent application Ser. No.
08/030,644 filed Mar. 12, 1993, the disclosures of which are hereby
incorporated by reference. Other useful BPI protein products
include peptides based on or derived from BPI which are described
in co-owned and co-pending U.S. patent application Ser. No.
08/274,299 filed Jul. 11, 1994, by Horwitz et al. and U.S. patent
application Ser. No. 08/273,540, filed Jul. 11, 1994, by Little et
al.
[0028] Presently preferred BPI protein products include
recombinantly-produced N-terminal fragments of BPI, especially
those having a molecular weight of approximately between 21 to 25
kD such as rBPI.sub.21 or rBPI.sub.23, dimeric forms of these
N-terminal fragments. Additionally, preferred BPI protein products
include rBPI.sub.50 and BPI-derived peptides.
[0029] The administration of BPI protein products is preferably
accomplished with a pharmaceutical composition comprising a BPI
protein product and a pharmaceutically acceptable diluent,
adjuvant, or carrier. The BPI protein product may be administered
without or in conjunction with known surfactants, other
chemotherapeutic agents. A preferred pharmaceutical composition
containing BPI protein products comprises the BPI protein product
at a concentration of 1 mg/ml in citrate buffered saline (5 or 20
mM citrate, 150 mM NaCl, pH 5.0) comprising 0.1% by weight of
poloxamer 188 (Pluronic F-68, BASF Wyandotte, Parsippany, N.J.) and
0.002% by weight of polysorbate 80 (Tween 80, ICI Americas Inc.,
Wilmington, Del.). Another preferred pharmaceutical composition
containing BPI protein products comprises the BPI protein product
at a concentration of 2 mg/ml in 5 mM citrate, 150 mM NaCl, 0.2%
poloxamer 188 and 0.002% polysorbate 80. Such preferred
combinations are described in co-owned, co-pending, U.S. patent
application Ser. No. 08/190,869 filed Feb. 2, 1994 (McGregor et
al., "Improved Pharmaceutical Compositions"), and U.S. patent
application Ser. No. 08/012,360 filed Feb. 2, 1993 (McGregor et
al., "Improved Pharmaceutical Composition"), the disclosures of
which are incorporated herein by reference.
[0030] The BPI protein product can be administered by any known
method, such as orally, systemically (such as by intravenous,
intramuscular or other injection), or as an aerosol. Medicaments
can be prepared for oral administration or by injection or other
parenteral methods and preferably include conventional
pharmaceutically acceptable carriers and adjuvents as would be
known to those of skill in the art. The medicaments may be in the
form of a unit dose in solid, semi-solid and liquid dosage forms
such as tablets, pills, powders, liquid solutions or suspensions,
and injectable and infusible solutions. Effective dosage ranges
from about 100 .mu.g/kg to about 10 mg/kg of body weight are
contemplated. Intravenous administration is a preferred method for
treatment of leprosy.
[0031] It is contemplated that aerosol administration to the lungs
will be a preferred method for treating other Mycobacterial
infections, such as tuberculosis. Such aerosol formulations would
be manufactured by means that are known in the art, and
administered by metered-dose inhaler, updraft nebulization, or
other means known in the art.
[0032] An aspect of the present invention is to provide methods of
treating a subject suffering from any of the physiological effects
of Mycobacterial LAM. As described above, the physiological effects
of LAM depend on a number of factors, including the source and
concentration of the LAM, and the length of time to which host
cells are exposed to LAM. Example 3, infra, demonstrates that
20-100 .mu.g/ml of nonviable, desiccated M. tuberculosis added to
whole blood will stimulate TNF production by the monocytes in the
blood. Other studies described above have shown that 50-100
.mu.g/ml of LAM will down-regulate macrophage functions and
expression (TNF, and the like) and prevent macrophage activation,
said methods comprise administering a BPI protein product to the
subject. Methods are provided for treating a subject suffering from
the effects of increased cytokine production caused by the
physiological presence of LAM. Methods are also provided for
treating a subject suffering from LAM-induced inhibition of
macrophage activation, and the effects thereof. Methods and
formulations by which a BPI protein product may be administered,
including preferred methods and formulations, are the same as those
set forth above for the treatment of Mycobacterial infection.
[0033] Because of the harmful physiological effects that
Mycobacterial LAM can have on a subject, even in the absence of
viable Mycobacteria, methods are provided in the present invention
by which a fluid containing LAM may be decontaminated prior to
administration of the fluid into a subject. Such methods comprise
contacting the fluid with a BPI protein product prior to
administration, under conditions such that LAM forms a complex with
the BPI protein product, thereby decontaminating the fluid. By way
of nonlimiting examples, such methods may be applied to fluids such
as blood, plasma, blood serum, bone marrow, isotonic solutions,
pharmaceutical agents, or cell culture reagents.
[0034] BPI protein product is thought to interact with a variety of
host defense elements present in whole blood or serum, including
complement and LBP, and other cells and components of the immune
system. Such interactions might result in potentiating and/or
synergizing the anti-microbial activities. Because of these
interactions, BPI protein products are expected to exert even
greater activity in vivo than in vitro. Thus, while in vitro tests
are predictive of in vivo utility, absence of activity in vitro
does not necessarily indicate absence of activity in vivo. For
example, BPI has been observed to display a greater bactericidal
effect on certain gram-negative bacteria in whole blood or plasma
assays than in assays using conventional media. [Weiss et al., J.
Clin. Invest. 90: 1122-1130 (1992)]. This may be because
conventional in vitro systems lack the blood elements that
facilitate or potentiate BPI's function in vivo, or because
conventional media designed to maximize bacterial growth contain
higher than physiological concentrations of magnesium and calcium,
inhibitors of BPI protein product antibacterial activity.
[0035] Therapeutic effectiveness is based on a successful clinical
outcome, and does not require that an anti-mycobacterial agent or
agents kill 100% of the orgainism involved in the infection.
Frequently, reducing organism load by one log (factor of 10)
permits the host's own defenses to control the infection. In
addition, augmenting an early anti-mycobacterial effect can be
particularly important in addition to any long-term
anti-mycobacterial effect. These early events are a significant and
critical part of therapeutic success, because they allow time for
host defense mechanisms to activate.
[0036] It is also contemplated that the BPI protein product be
administered with other products that potentiate the
anti-mycobacterial activity of BPI protein products. For example,
serum complement potentiates the gram-negative bactericidal
activity of BPI protein products; the combination of BPI protein
product and serum complement provides synergistic
bactericidal/growth inhibitory effects. See, e.g., Ooi et al. J.
Biol. Chem., 265: 15956 (1990) and Levy et al. J. Biol. Chem., 268:
6038-6083 (1993) which address naturally occurring 15 kD proteins
potentiating BPI antibacterial activity. See also co-owned,
co-pending U.S. patent application Ser. No. 08/093,201 filed Jul.
14, 1993, and continuation-in-part, U.S. patent application Ser.
No. 08/274,303 filed Jul. 11, 1994 which describes methods for
potentiating gram-negative bactericidal activity of BPI protein
products by administering lipopolysaccharide binding protein (LBP)
and LBP protein products. The disclosures of these applications are
incorporated by reference herein. LBP protein derivatives and
derivative hybrids which lack CD-14 immunostimulatory properties
are described in co-owned, co-pending U.S. patent application Ser.
No. 08/261,660, filed Jun. 17, 1994 as a continuation-in-part of
U.S. patent application Ser. No. 08/079,510, filed Jun. 17, 1993,
the disclosures of which are incorporated by reference herein.
[0037] An aspect of this invention includes the use of a
composition comprising a BPI protein product for the manufacture of
a medicament for the therapeutic application of treating any of the
aforementioned conditions or diseases from which a subject suffers.
The medicament may include, in addition to a BPI protein product,
other chemotherapeutic agents such as known anti-mycobacterial
antibiotics or surfactants. The medicament may additionally or
alternatively include one or more additional pharmaceutically
acceptable components, such as diluents, adjuvants, or
carriers.
[0038] An aspect of the present invention is the ability to provide
more effective treatment of Mycobacterial infection by virtue of
the synergistic increase in or potentiation of the anti-bacterial
activities of an anti-Mycobacterial antibiotic or BPI protein
product. As previously noted, anti-Mycobacterial antibiotic therapy
currently involves administration of one or more (and frequently
three or more) antibiotics such as isoniazid, rifampin, ethambutol,
p-aminosalicylic acid, pyrazinamide, streptomycin, capreomycin,
cycloserine, ethionamide, kanamycin, amikacin, amithiozone,
rifabutin, clofazimine, arithromycin, clarithromycin, ciprofloxacin
and ofloxacin. Unlike some therapeutic agents, BPI protein product
is easily administered and produces no inflammatory reaction. An
aspect of the present invention is the ability to treat
Mycobacterial organisms that are normally resistant to one or more
antibiotics. A further aspect is the ability to use lower
concentrations of relatively toxic or expensive antibiotics such as
rifampin. Because the use of some antibiotics is limited by their
systemic toxicity or prohibitive cost, lowering the concentration
of antibiotic required for therapeutic effectiveness reduces
toxicity and/or cost of treatment, and thus allows wider use of the
antibiotic. The present invention may also provide quality of life
benefits due to, e.g., decreased duration of therapy, reduced stay
in intensive care units or overall in the hospital, with the
concomitant reduced risk of serious nosocomial (hospital-acquired)
infections.
[0039] The invention further provides pharmaceutical compositions
for treatment of Mycobacterial infection and the sequelae thereof
comprising the combination of a BPI protein product and an
antibiotic which is present in an amount effective to have
synergistic or potentiating bactericidal/bacteriostatic properties,
including increased susceptibility or reversal of resistance. The
pharmaceutical composition can comprise a
pharmaceutically-acceptable diluent, adjuvant or carrier.
[0040] Methods of the present invention are more fully illustrated
by the nonlimiting examples which follow. Example 1 address BPI
protein products binding to a species of Mycobacterium, M.
tuberculosis. Example 2 address prospective use of BPI protein
products in binding purified LAM of Mycobacteria. Examples 3 and 4
describe attempts to reverse Mycobacteria-induced cytokine
production in whole human blood. Example 5 addresses use of BPI
protein products in combination with anti-mycobacterial antibiotics
to inhibit M. tuberculosis growth. Remaining Examples 6-13 address
prospective in vitro and in vivo use of BPI protein products
according to methods of this invention. The models described in
those examples and/or other models known in the art are used to
predict the efficacy and the optimal BPI protein product
formulations of the methods of invention.
EXAMPLE 1
[0041] An enzyme linked immunosorbent assay (ELISA) was conducted
to determine binding of a BPI protein product to M. tuberculosis.
Specifically, non-viable, desiccated M. tuberculosis H37 RA (Difco,
Detroit, Mich.) was suspended in DPBS (25 .mu.g/ml) and used to
coat microtiter wells overnight at 37.degree. C. Wells were also
coated with either 25 .mu.g/ml Lipid A (E. coli J5 mutant, RIBI,
Hamilton, Mont.) or 500 .mu.l DPBS to demonstrate the functionality
and specificity of rBPI.sub.23. After washing (3.times. with
DPBS+0.05% Tween 20), the plates were blocked for 1 hr. at room
temperature with 200 .mu.l/well of DPBS+1% non-fat milk. After
washing as above, 50 .mu.l solutions of either various
concentrations of rBPI.sub.23 (in DPBS containing 0.05% Tween 20)
or DPBS (negative control) were added to the wells, which were then
incubated for 1 hr. at 37.degree. C. The wells were again washed as
above, and the amount of rBPI.sub.23 bound to the wells was
determined using an anti-rBPI.sub.23 mouse monoclonal antibody
(designated .alpha.BPI MAb-24) and an enzyme conjugated anti-murine
IgG antibody (HRP-Ab, Zymed #61-0120, San Francisco, Calif.). To
each well 100 .mu.l of .alpha.BPI MAb-2-4 was added (100 ng/ml in
DPBS+0.05% Tween 20), and the plates were incubated 1 hr. at
37.degree. C. After washing as above, 100 .mu.l of HRP-Ab was added
(1:1000 in DPBS+0.05% Tween 20) to each well and the plates were
again incubated 1 hr. at 37.degree. C. After washing the plates as
above, 100 .mu.l substrate in 0.1M citrate plus 1:50 ABTS (20 mg/ml
stock) and 1:1000H.sub.2O.sub.2 was added to each well. The plates
were incubated 10-30 min. at room temperature, and absorbance
readings were taken at 405 nm (OD 405).
[0042] The results of the experiment are represented graphically in
FIG. 1, which depicts the ability of varying concentrations
rBPI.sub.23 to bind to J5 Lipid A (filled triangles); to M.
tuberculosis (open squares); and to the no antigen-free control
(filled circles). The abscissa of each measurement represents the
concentration of rBPI.sub.23, and the ordinate represents the
average OD 405 measurements from four trials. Error bars reflect
the variation in OD 405 readings for each data point.
[0043] This experiment demonstrated that rBPI.sub.23 binds
specifically to non-viable desiccated M. tuberculosis. The
functionality of the rBPI.sub.23 used in these experiments was
confirmed by the results of the Lipid A (positive control) binding
assay, and the specificity of the experiments was confirmed by the
lack of binding to the negative control samples.
EXAMPLE 2
[0044] In this example, an ELISA Assay is conducted to determine
binding of a BPI protein product to the lipoarabinomannan portion
of Mycobacteria. The binding activity of BPI protein product (e.g.,
rBPI.sub.23) to LAM is demonstrated as described in the previous
example, except LAM purified from a species of Mycobacterium,
(e.g., M. tuberculosis or M. leprae) is substituted for the
nonviable M. tuberculosis used to coat the ELISA plates in that
example. Purified LAM is isolated as described by Hunter et al., J.
Biol. Chew., 261: 12345-12351 (1986). Specific binding of
biologically active BPI protein product is demonstrated by
comparison of the OD 405 readings from the LAM coated wells with
positive and negative controls.
EXAMPLE 3
[0045] The following experiment was conducted to determine the
effect of a BPI protein product, rBPI.sub.23, on
Mycobacteria-induced cytokine production in whole human blood.
Whole human blood from healthy volunteers was collected into
Vacutainer tubes (ACD, Beckton Dickinson, Rutherford, N.J.).
Aliquots of blood (225 .mu.l) were mixed with either rBPI.sub.23
(10 .mu.g/ml final) or the protein thaumatin (10 .mu.g/ml final in
5 ml) as a negative control. RPMI medium (20 .mu.l) was added to
each sample. Varying dilutions (0-8 ng/ml) of either E. coli O113
LPS (Ribi, Hamilton Mich.) or of non-viable, desiccated M.
tuberculosis H37 RA (0-100 .mu.g/ml) (Difco, Detroit Mich.) were
added to the samples, which were then incubated at 37 C for 6
hours. The reactions were stopped by the addition of 750 .mu.l of
RPMI medium, the samples were centrifuged at 500 g for 7 min, and
stored at -20.degree. C. until analyzed. The supernatant was
assayed for cytokine (TNF) levels based on a standard curve,
according to the manufacturers' recommendation (Biokine ELISA test,
T Cell Sciences, Cambridge, Mass.).
[0046] The assay results revealed that rBPI.sub.23 at 10 .mu.g/ml
had no inhibitory effect on M. tuberculosis-induced TNF release at
the concentration (20-100 .mu.g/ml) of M. tuberculosis added to the
blood samples. The same concentration of rBPI.sub.23 eliminated
LPS-induced TNF release at the LPS concentrations tested (2-8
ng/ml). The lack of inhibitory effect on cytokine induction by M.
tuberculosis may be the result of use of sub-optimal dosage levels.
Alternatively, some component of the Mycobacterial cell wall other
than the LAM bound by rBPI.sub.23 may be responsible for inducing
cytokine production at the Mycobacterium concentrations tested.
EXAMPLE 4
[0047] In this example, multiple additional assays were conducted
to assess the inhibitory effect of BPI protein products on
mycobacterial (M. tuberculosis or M. smegmatis) induced production
of tumor necrosis factor (TNF) by monocytes/macrophages present in
whole human blood. Briefly summarized, live or heat killed
mycobacteria at varying concentrations was added to whole blood and
incubated with either a fixed amount BPI protein product or acetate
buffer negative control solution. After incubation, the content of
TNF present was assessed by standard means. The TNF content of BPI
protein product treated samples was then compared to the TNF
content of buffer control samples to determine the relative
inhibitory effect of the BPI protein product tested. Whole blood
samples were obtained from healthy human volunteers and aliquoted
as in Example 3. To each tube containing 225 .mu.l of whole blood
was added from 0 to 1.times.10.sup.7 live or heat killed
mycobacteria. Depending on whether one or two BPI protein products
were to be tested, four or six tubes were prepared at each
concentration of bacteria, after which the tubes were incubated at
37.degree. C. for 15 minutes. To two of the tubes at each bacterial
concentration was added either a selected BPI protein product at a
final concentration of 4 .mu.g/ml or acetate buffer (as a negative
control), after which is tubes were further incubated at 37.degree.
C. for 5 to 6 hours. Thereafter 750 .mu.l of RPMI 1640 was added to
each sample and the samples were centrifuged at 17,000 rpm (500 g?)
for 6 minutes. Supernatants were stored at -70.degree. C. until
thawed immediately prior to testing for TNF content using the
Biokine ELISA test kit as in Example 3.
[0048] In a first series of assays, heat-killed M. tuberculosis
(strain H37Ra) was employed as the TNF stimulating organism. In a
first test on whole blood, no substantial increased in TNF levels
was observed until bacteria were added at a concentration of
1.times.10.sup.6 organisms and the presence of rBPI.sub.23 in the
samples resulted in an approximately 70% inhibition of TNF
production. In a second test involving two separate whole blood
assays, inhibitory effects of both rBPI.sub.23 and BPI holoprotein
were assessed. Substantial increases in TNF concentration over
basal levels (no microorganisms added) were observed commencing at
microorganism concentrations of 3.times.10.sup.5 up through
1.times.10.sup.7. Overall, inhibition of TNF production by 20% or
greater was observed when rBPI.sub.23 was added at all such
organism levels. Lesser degrees of inhibition were noted for the
BPI holoprotein (with no inhibition at all noted in one duplicate
test at the highest concentration of organisms). The lesser effects
of the holoprotein in these assays are likely attributable to the
lower molar concentration employed. A third M. tuberculosis test
was performed on whole blood drawn from four different volunteers,
using rBPI.sub.23 as the test compound. Expectedly, the level of
inhibition of TNF formation by the uniform dose of BPI protein
product varied from subject to subject. With the exception of one
subject's blood samples (wherein inhibition was observed only at
intermediate microorganism concentration of 1.times.10.sup.6 and
not at all at concentrations of 1.times.10.sup.7), the BPI protein
product provided for at least about 10% and up to about 50% TNF
inhibition, with higher inhibitory levels being observed at higher
microorganism concentrations. A fourth test involving M.
tuberculosis was carried out using rBPI.sub.23 at 4 .mu.g/ml and 8
.mu.g/ml concentrations. The BPI protein product was again observed
to inhibit TNF at the 4 .mu.g/ml level, with the greatest effects
being observed at microorganism concentrations of 3.times.10.sup.6.
Doubling the concentration of test compound to 8 .mu.g/ml did not
enhance, and in fact somewhat diminished, inhibitory effects
observed.
[0049] In a second series of assays, heat killed M. smegmatis was
employed to stimulate TNF production in whole blood. In a first
test, whole blood from eight different patients was employed and
was subjected to contact with concentrations of 0,
0.5.times.10.sup.5, 1.times.10.sup.5, 1.times.10.sup.6 and
1.times.10.sup.7 organisms. FIG. 2 provides a graphic
representation of the sum of the results observed and indicated
that the rBPI.sub.23 product tested at 4 .mu.g/ml was an effective
inhibitor of TNF production at all bacterial concentrations. In a
second test involving blood from two different subjects, TNF
production inhibitory effects of rBPI.sub.23 were assessed for M.
smegmatis, E. coli and S. aureus. Expectedly, significant TNF
inhibitory effects were observed in the E. coli treated blood, with
the greatest present with microorganism concentrations of
1.times.10.sup.5 and the lesser effects at higher concentrations of
organisms. Similarly, no substantial TNF inhibitory effects were
observed for the BPI protein product in the S. aureus assay.
Variable results were seen in the M. smegmatis assay; pronounced
inhibitory effects were observed in one subject's blood at the
1.times.10.sup.7 concentration of organisms, while inhibition was
observed in the other blood sample only at the 1.times.10.sup.6
microorganism concentration.
[0050] The above assay results demonstrate in vitro effectiveness
of BPI protein products in inhibiting induction of tumor necrosis
factor by mycobacterial species and are predictive of in vivo
efficacy in human patients.
EXAMPLE 5
[0051] In this example, rBPI.sub.23 at varying concentrations was
assessed for its growth inhibitory effect on M. tuberculosis
treated with varying concentrations of the anti-Mycobacterial
antibiotics isoniazid (INH) and rifampin (RMP). Briefly summarized,
pure cultures of M. tuberculosis (MTB) were incubated for 24 hours
with varying concentrations of rBPI.sub.23 and antibiotic. Cultures
were added to Bactec.RTM. bottles (Johnston Laboratories,
Cockeysville, Md.) containing .sup.14C labeled nutrients and daily
"growth index" values were determined accordingly to the supplier's
instructions on the basis of .sup.14CO.sub.2 evolved from the
medium. In separate assays, concentrations of rBPI.sub.23 of 0,
3.9, 15.6, 62.5, 250 and 1000 .mu.g/ml were combined with INH at
levels of 0, 0.006, 0.012, 0.025 and 0.05 .mu.g/ml or RMP levels of
0, 0.12, 0.25, 0.5 and 1.0 .mu.g/ml. Growth index values were
assessed daily starting the second day after inoculation into the
vials through to the eighteenth day. In the INH assay, no BPI
protein product was added, growth index values characteristically
gradually increased over time and as a function of the dosage of
antibiotic employed (increases generally began earlier and rose
more steeply at lower doses than at higher doses). Addition of
rBPI.sub.23 had variable effects in enhancing or diminishing
antibiotic effects on growth index values, depending on the
concentration employed. An intermediate dose (62.5 .mu.g/ml) of BPI
protein product consistently tended to reduce growth index values
at all doses of INH tested and thus operated to enhance INH growth
inhibitory effects. Similar but less pronounced enhancement effects
were observed for the 15.6 .mu.g/ml rBPI.sub.23 dose. Lower (0 and
3.9 .mu.g/ml) and higher (250 and 1000 .mu.g/ml) doses of the BPI
protein product generally diminished the antibiotic effects of INH,
with the highest rBPI.sub.23 dose invariably functioning to
increasing "swamp out" INH effects on growth index toward the
middle of the test period. At later times in the test period,
however, the highest doses of BPI protein product appeared to
suppress and actually reverse the above-noted characteristic
increases in growth index over time. In combinative assay with RMP,
rBPI.sub.23 had no discernible enhancing effect at the highest
doses of the antibiotic 0.5 and 1.0 .mu.g/ml where there was
essentially no increase in growth index throughout the entire test
period. At lesser concentrations pf RMP, there tended to be a
dose-dependent enhancement effect of the BPI protein product, with
the greatest degree of enhancement occurring at the highest doses
of rBPI.sub.23 and no evidence of the "swamp out" effects observed
for combinations with INH intermediate times within the test
period.
[0052] The results set out above establish utility of BPI protein
product concurrently filed in enhancing the growth inhibitory
effects of anti-mycobacterial antitiotics.
EXAMPLE 6
[0053] The following experiment is conducted to determine the in
vitro inhibitory effect of a BPI protein product on the growth of a
Mycobacterium species, Mycobacterium tuberculosis (MTB). The
procedure can be performed with other cultivable Mycobacterial
species and employs concentrations of a BPI protein product that
would be readily generated in human serum by ordinary modes of oral
or parenteral administration and/or readily delivered to lung
surface by aerosol administration. The effects of the BPI protein
product can be evaluated with and without non-ionic surfactants,
and/or standard antibiotics.
[0054] Log phase cultures of antibiotic-sensitive and
antibiotic-resistant MTB are incubated in either 7H11 broth medium
or whole human blood, to which the following is added: (a) nothing;
(b) surfactant; (c) standard MTB antibiotic; (d) antibiotic plus
surfactant. Cultures are incubated with varying concentrations of,
e.g., rBPI.sub.23. Duplicate cultures grown in each medium are also
left untreated by rBPI.sub.23 as a negative control. The organisms
are placed in Bactec.RTM. bottles (Johnston Laboratories,
Cockeysville, Md.) containing .sup.14C labeled nutrients.
rBPI.sub.23 challenged M. tuberculosis growth is determined by
measuring the elution of .sup.14CO.sub.2 from the medium, compared
to the appropriate negative control. The absence of the formation
.sup.14CO.sub.2 by the treated cultures is indicative of the
inhibitory affects of rBPI.sub.23 to MTB. Differential amounts of
.sup.14CO.sub.2 formed in the absence or presence of standard MTB
antibiotics and/or surfactants is indicative of the synergistic or
additive effect that a BPI protein product has when used
conjunctively with such agents. By comparing the results of this
experiment performed with varying concentrations of the BPI protein
product, the effective concentration of the BPI protein product is
optimized. Radiometric assays to test the susceptibility of
Mycobacterial species to drugs have been described previously. See
McClatchy (cited supra) and references therein.
EXAMPLE 7
[0055] The following experiment is conducted to determine the in
vitro effects of a BPI protein product (rBPI.sub.23) in an M.
leprae model. A palmitic acid oxidation assay is used to measure
the "viability" of the uncultivable leprosy bacillus adhered to
filter paper and "grown" in a .sup.14C-palmitic acid-containing
medium. In this method .sup.14CO.sub.2 evolved from the metabolism
by M. leprae of .sup.14C-palmitic acid is trapped on filter paper
moistened with NaOH and radioactivity is determined with a liquid
scintillation counter. Susceptibility to BPI protein product
formulations is determined by differences in radioactivity for M.
leprae tested with such formulations and treated control
cultures.
EXAMPLE 8
[0056] The following experiment, which is a variation of an assay
conducted by Mittal et al., J. Clin. Microbiol., 17 (4): 704-707
(1983), is conducted to determine the in vitro inhibitory effect of
BPI protein product on the growth of Mycobacterium leprae. The
effects of different concentrations of BPI protein product on M.
leprae are evaluated with and without non-ionic surfactants, and/or
standard antibiotics. The procedures as described by Mittal et al.
are outlined below.
[0057] Skin biopsy specimens from lepromatous patients are
homogenized and are used to inoculate suspensions of mouse
peritoneal macrophages cultured in RPMI 1640 (GIBCO Biocult,
Irvine, Scotland) enriched with 30% fetal calf serum. After
incubating 18 hours, fresh media containing
[methyl-.sup.3H]-thymidine (Amersham International Ltd., Arlington
Heights, Ill.) is added and the cultures are incubated for 14 days.
The procedure of Mittal et al. is varied by testing the effect of
different concentrations of BPI protein product with or without
surfactants and/or antibiotics on .sup.3H-thymidine incorporation.
Macrophages containing phagocytosed viable M. leprae will
incorporate .sup.3H-thymidine at a 2 to 10-fold higher rate than
control cultures containing heat killed M. leprae. Greater than 50%
inhibition of .sup.3H-thymidine-incorporation is indicative of
bactericidal efficacy of the test product.
EXAMPLE 9
[0058] An experiment is conducted to determine the in vivo effect
that a BPI protein product will have on M. tuberculosis species.
The model employed is a variation of that used by Lalande et al.,
Antimicrobial Agents and Chemotherapy, 37 (3): 407-413 (1993), to
assess the efficacy of antimicrobial agents against M.
tuberculosis. Mice inoculated intravenously with M. tuberculosis
are treated with various BPI protein product doses alone or in
combination with surfactants and/or antibiotics. The efficacy of
such treatment regiments is analyzed as described.
EXAMPLE 10
[0059] The following experiment is conducted to determine the
effect that a BPI protein product will have on M. leprae in vivo.
The model to be used is a variation of that developed by Shepard to
study the effect of compounds on the growth of M. leprae in the
footpads of infected mice. Shepard et al., Proc. Soc. Exp. Biol.
Med., 109: 636-638 (1962); Shepard, J. Exp. Med. 112: 445-454
(1960). Briefly, leprosy bacilli are inoculated into foot-pads of
mice, which are subsequently treated with different amounts of test
compound with or without known antibiotics and/or surfactants.
Untreated infected mice are used as a control. Mice from each
treatment regimen are sacrificed at monthly intervals, and sections
cut from the infected foot. The presence of an area containing
acid-fast bacteria can be observed microscopically and/or the
number of such bacteria can be counted. See Shepard and McRae, Int.
J. Lepr., 36: 78-82. Differences between M. leprae bacteria levels
observed in treated versus control mice is indicative of the
bacteriostatic or bactericidal efficacy of a given BPI treatment
regimen. The metabolic status of isolated M. leprae may also be
measured. Franzblau and Hastings, Antimicrobial Agnes and
Chewmotherapy, 31 (5): 780-783 (1987).
EXAMPLE 11
[0060] The following experiments are designed to demonstrate that
BPI protein product is able to inhibit the ability of low
concentration of LAM to induce cytokines, yet reverse the
unresponsive state that attends higher concentrations of LAM.
Increasing concentrations of LAM are pretreated with BPI protein
product at varying concentrations. These complexes are applied to
peritoneal macrophages from normal and Mycobacterium species
infected mice. TNF production by treated cells will be
assessed.
EXAMPLE 12
[0061] A variation of the armadillo model developed by Kirchheimer
et al., Int. J. Lepr., 39: 693-702 (1971); Id., 40: 229 (1972), is
employed to study the in vivo effect of BPI protein product test
compositions on the growth of M. leprae in infected armadillos.
Briefly, leprosy bacilli are inoculated into armadillos, which are
subsequently treated with different amounts of a test composition.
The test compositions will comprise a BPI protein product, e.g.
rBPI.sub.23, with or without known antibiotics and/or surfactants.
Untreated infected specimens are used as a control. Armadillos from
each treatment regimen are examined and biopsy specimens analyzed
by procedures known in the art. M. leprae isolated from armadillos
is assayed for metabolic activity. Differences between the
appearance of lesions, differences in M. leprae bacterial
concentrations, and differences in the metabolic activity of M.
leprae isolates in treated versus control specimens are indicative
of the bacteriostatic or bactericidal efficacy of a given BPI
treatment regimen.
EXAMPLE 13
[0062] The following experiment is conducted to determine the level
of decontamination of a fluid containing LAM that can be achieved
by treatment with a BPI protein product. Whole human blood, plasma,
blood serum or the like is passed through a column containing a
matrix, to which a BPI protein product is bound. Such matrix may be
constructed by any means known to those skilled in the art. LAM in
the fluid complexes with the BPI protein product affixed to the
matrix as the fluid is passed through the column. The absence of
LAM in the fluid eluted from the column demonstrates the
effectiveness of a BPI protein product at decontaminating a fluid
containing LAM.
[0063] Alternatively, monoclonal antibodies with binding
specificity for a BPI protein product, such as the antibodies
employed in Example 1, are affixed to the matrix. A sufficient
amount of a BPI protein product is added to the mixture to bind any
LAM present in the fluid. The fluid is purified by passing it
through the column. The .alpha.BPI antibodies affixed to the column
bind the LAM/BPI protein product complex in the fluid, and the
fluid eluted from the column is analyzed for the presence or
absence of LAM contamination.
[0064] Numerous modifications and variations in the practice of the
invention are expected to occur to those skilled in the art upon
consideration of the foregoing description of the presently
preferred embodiments thereof. For example, while the above
illustrative examples principally address studies predictive of
antibacterial effects in the context M. tuberculosis and M. leprae,
model studies of infection with, e.g., M. avium [see, e.g., Brown
et al., Antimicrob. Agents and Therapy, 37 (3): 398-402 (1993)] are
also expected to reveal effectiveness of BPI protein product
therapies. As another example, preliminary experimental data
indicates that BPI protein products alone and/or in combination
with cytokines such as gamma interferon (and in combination with
antibiotics as well) can enhance the rate at which human monocytes
phagocytize Mycobacterial organisms. Combinative therapies
involving administration of cytokines along with BPI protein
products (and antibiotics) are thus within the scope of the
invention). Consequently, the only limitations which should be
placed upon the scope of the present invention are those which
appear in the appended claims.
Sequence CWU 1
1
* * * * *