Therapeutic uses of N-terminal BPI protein products in ANCA-positive patients

Abstract

Improved therapeutic uses for N-terminal BPI protein products are described in patients that have BPI-reactive anti-neutrophil cytoplasmic antibodies.

Description

BACKGROUND OF THE INVENTION

[0001] The present invention relates generally to novel improved methods of treating subjects that have BPI-reactive anti-neutrophil cytoplasmic antibodies by administering N-terminal bactericidal/permeability-increasing protein (BPI) protein products.

[0002] Anti-neutrophil cytoplasmic antibodies (ANCA) have been recognized as a class of autoantibodies that react with the cytoplasmic constituents of neutrophils and monocytes. ANCA are detected by indirect immunofluorescence (IIF) on ethanol-fixed neutrophils, and produce at least three distinct immunofluorescence patterns: cANCA (cytoplasmic, or ‘classic’ pattern), pANCA (perinuclear to nuclear pattern) or AANCA (atypical, with a peculiar “snow drift pattern”). [Kallenberg et al., Am. J. Med., 93:675-682 (1992).] The presence of ANCA has been associated with various idiopathic systemic vasculitis disorders (i.e., inflammation of and damage to the blood vessels) and with other inflammatory disorders, and can be diagnostic of certain vasculitides. These vasculitides are sometimes called ANCA-associated vasculitides (AAV). A pathophysiologic role for ANCA in vasculitides has been proposed but remains to be definitively established. [Kallenberg et al., Clin. Exp. Immunol., 100:1-3 (1995).]

[0003] The antigen primarily recognized by c-ANCA proved to be a 29 kd serme protease from myeloid azurophilic granules known as proteinase 3 (PR-3). The presence of anti-PR3 is highly correlated to cANCA and is specific for idiopathic vasculitides such as Wegener's granulomatosis (WG), microscopic polyarteritis (MPA) and the renal limited pauci-immune necrotizing and crescentic glomerulonephritis (NCGN). [Kallenberg, 1995, supra.]

[0004] One of the antigens recognized by p-ANCA is myeloperoxidase (MPO), another constituent of azurophilic granules. However, only a minority of p-ANCA-positive sera reacts with MPO. Anti-MPO antibodies have been found to be specific for systemic vasculitis and idiopathic crescentic glomerulonephritis. Anti-MPO antibodies are also found in patients with anti-glomerular basement membrane disease and in some sera of patients with systemic lupus erythematosus (SLE). The presence of p-ANCA has also been described in sera from patients with a wide range of different disorders such as colitides (including ulcerative colitis, inflammatory bowel disease, Crohn's disease and collagenous colitis), autoimmune liver diseases (including autoimmune chronic active hepatitis, primary sclerosing cholangitis and primary biliary cirrhosis) and rheumatoid arthritis. [Kallenberg, 1992, supra.] These latter p-ANCA generally do not react with MPO, and their antigenic specificities are largely unknown. Some antibodies to leukocyte elastase and lactoferrin have also been described that produce a p-ANCA pattern by IIF. Antibodies to elastase occur occasionally in sera from patients with vasculitis or drug-induced systemic autoimmune disease. Lactoferrin antibodies have been seen in a few patients with vasculitis, primary sclerosing cholangitis and ulcerative colitis, and in a minority of patients with rheumatoid arthritis. Their diagnostic value has not been established yet.

[0005] BPI has been identified as another ANCA antigen. Falk et al., Third Int'l Workshop on ANCA, Am J. Kidney Dis., 18:197 (abst. 6) (1991), reported that 11 of 51 cANCA-positive samples recognized a 57-kD antimicrobial cationic protein (CAP57), which was later identified as BPI by N-terminal amino acid sequence homology and immunoreactivity. This CAP57-specific cANCA staining was blocked by anti-CAP57 monoclonal antibodies but not by anti-MPO or anti-PR3 monoclonal antibodies. No clinical details of the patients, with respect to whether or not they had vasculitis, and if so, the distribution, were reported in this abstract.

[0006] Zhao et al., Clin. Exp. Immunol., 99:49-56 (1995) also reported the identification of BPI-reactive ANCA in serum samples from patients with suspected vasculitis. Of 100 historical serum samples that were double-negative for PR3 and MPO specificity by EUSA, 45% were reactive with purified BPI. Of 400 newly obtained samples sent for routine ANCA testing, 11% had BPI specificity, suggesting that BPI is an important ANCA antigen. The PR3 and MPO specificities in these 400 new samples were 10/400 and 14/400 respectively. Zhao et al. conjectured that these human autoantibodies against BPI might block the bactericidal and LPS-neutralizing activities of BPI, allowing these non-neutralized products to directly cause vascular damage and initiate vasculitis.

[0007] BPI is a protein isolated from the granules of mammalian polymorphonuclear leukocytes (PMNs or neutrophils), which are blood cells essential in the defense against invading microorganisms. Human BPI protein has been isolated from PMNs 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)]. BPI obtained in such a manner is referred to herein as natural BPI and has been shown to have 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 and the nucleic acid sequence of DNA encoding the protein have been reported in figure 1 of Gray et al., J. Biol. Chem., 264:9505 (1989), incorporated herein by reference. The Gray et al. amino acid sequence is set out in SEQ ID NO: 1 hereto. U.S. Pat. No. 5,198,541 discloses recombinant genes encoding and methods for expression of BPI proteins, including BPI holoprotein and fragments of BPI.

[0008] BPI is a strongly cationic protein. The N-terminal half of BPI accounts for the high net positive charge; the C-terminal half of the molecule has a net charge of −3. [Elsbach and Weiss (1981), supra.] A proteolytic N-terminal fragment of BPI having a molecular weight of about 25 kD possesses essentially all the anti-bacterial efficacy of the naturally-derived 55 kD human BPI holoprotein. [Ooi et al., J. Bio. Chem., 262: 14891-14894 (1987)]. In contrast to the N-terminal portion, the C-terminal region of the isolated human BPI protein displays only slightly detectable anti-bacterial activity against gram-negative organisms. [Ooi et al., J. Exp. Med., 174:649 (1991).] An N-terminal BPI fragment of approximately 23 kD, referred to as “rBPI23,” has been produced by recombinant means and also retains anti-bacterial activity against gram-negative organisms. [Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992).] An N-terminal analog of BPI, rBPI21, has been produced as described in Horwitz et al., Protein Expression Purification, 8:28-40 (1996).

[0009] The bactericidal effect of BPI has been reported to be highly specific to gram-negative species, e.g., in Elsbach and Weiss, Inflammation: Basic Principles and Clinical Correlates, eds. Gallin et al., Chapter 30, Raven Press, Ltd. (1992). The precise mechanism by which BPI kills gram-negative bacteria is not yet completely elucidated, but it is believed that BPI must first bind to the surface of the bacteria through electrostatic and hydrophobic interactions between the cationic BPI protein and negatively charged sites on LPS. In susceptible gram-negative bacteria, BPI binding is thought to disrupt LPS structure, leading to activation of bacterial enzymes that degrade phospholipids and peptidoglycans, altering the permeability of the cell's outer membrane, and initiating events that ultimately lead to cell death. [Elsbach and Weiss (1992), supra]. LPS has been referred to as “endotoxin” because of the potent inflammatory response that it stimulates, i.e., the release of mediators by host inflammatory cells which may ultimately result in irreversible endotoxic shock. BPI binds to lipid A, reported to be the most toxic and most biologically active component of LPS.

[0010] BPI protein products, as discussed infra, have a wide variety of beneficial activities in addition to their gram-negative bactericidal activities. The observation of antibodies reactive against BPI among ANCA-positive subjects suggests that these antibodies may interfere with the activities of BPI. A need therefore exists for improved methods of treating subjects that have BPI-reactive ANCA with BPI protein products.

SUMMARY OF THE INVENTION

[0011] The present invention provides novel improved methods of treating subjects that have non-N-terminal-BPI-reactive antibodies by administering N-terminal bactericidal/permeability-increasing (BPI) protein products. The invention is based on the discovery that BPI-reactive autoantibodies bind to BPI holoprotein but have very little reactivity with N-terminal BPI protein products. Interference with the beneficial activities of endogenous BPI or exogenous BPI protein products can therefore be avoided by administering N-terminal BPI protein products.

[0012] It is contemplated that these improved methods will be useful when the N-terminal BPI protein product is being administered for any of the indications presently known for BPI protein products. For example, the N-terminal BPI protein product may be administered to a human subject to ameliorate adverse effects associated with endotoxin in circulation, meningococcemia, hemorrhagic trauma, burn trauma, ischemia/reperfusion injury, or liver resection injury. A N-terminal BPI protein product may also be administered for the treatment of gram-negative bacterial infection, gram-positive bacterial or mycoplasmal infection, fungal infection, protozoal infection, chlamydial infection, mycobacterial infection, chronic inflammatory diseases, including rheumatoid and reactive arthritis, or to enhance the effectiveness of antibiotic activity, or to inhibit angiogenesis or to promote fibrinolysis.

[0013] Presently preferred N-terminal BPI protein products include amino-terminal fragments of BPI protein having a molecular weight of about 20 kD to 25 kD, rBPI23 or a dimeric form thereof, and rBPI21.

[0014] It is contemplated that the administration of BPI protein products, especially N-terminal BPI protein products, according to all aspects of the present invention may be accompanied by the concurrent administration of other therapeutic agents such as antimicrobial agents, including antibiotics and anti-fungal agents.

[0015] Numerous additional aspects and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of the invention which describes presently preferred embodiments thereof.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention provides improved methods of treating subjects that have non-N-terminal-BPI-reactive antibodies, the presence of which may interfere with the activities of BPI protein products in these subjects, by the administration of N-terminal BPI protein products. The invention is based on the discovery that BPI-reactive autoantibodies bind to BPI holoprotein but have very little reactivity with N-terminal BPI protein products; the ANCA-recognized epitopes thus appear to reside predominantly outside the N-terminal 193 amino acids of BPI.

[0017] BPI protein products are known to have a variety of beneficial activities. BPI protein products are known to be bactericidal for gram-negative bacteria, as described in U.S. Pat. Nos. 5,198,541 and 5,523,288, both of which are incorporated herein by reference. BPI protein products are also known to enhance the effectiveness of antibiotic therapy in gram-negative bacterial infections, as described in U.S. Pat. No. 5,523,288, which is incorporated herein by reference. BPI protein products are also known to be bactericidal for gram-positive bacteria and mycoplasma, and to enhance the effectiveness of antibiotics in gram-positive bacterial infections, as described in co-owned, co-pending U.S. application Ser. No. 08/372,783 filed Jan. 13, 1995, which is in turn a continuation-in-part of U.S. application Ser. No. 08/274,299 filed Jul. 11, 1994, and corresponding International Publication No. WO 95/08344 (PCT/US94/11225), all of which are incorporated herein by reference. BPI protein products are further known to exhibit anti-fungal activity, and to enhance the activity of other anti-fungal agents, as described in co-owned, co-pending U.S. application Ser. No. 08/372,105 filed Jan. 13, 1995, which is in turn a continuation-in-part of U.S. application Ser. No. 08/273,540 filed Jul. 11, 1994, and corresponding International Publication No. WO 95/19179 (PCT/US95/00498), and further as described for anti-fungal peptides in co-owned, co-pending U.S. application Ser. No. 08/621,259 filed Mar. 21, 1996, which is in turn a continuation-in-part of U.S. application Ser. No. 08/504,841 filed Jul. 20, 1994 and corresponding International Publication No. WO 96/08509 (PCT/US95/09262) and PCT Application No. PCT/US96/03845, all of which are incorporated herein by reference. BPI protein products are further known to exhibit anti-protozoan activity, as described in co-owned, co-pending U.S. application Ser. No. 08/273,470 filed Jul. 11, 1994 and corresponding International Publication No. WO 96/01647 (PCT/US95/08624), all of which are incorporated herein by reference. BPI protein products are known to exhibit anti-chlamydial activity, as described in co-owned, co-pending U.S. application Ser. No. 08/694,843 filed Aug. 9, 1996, all of which are incorporated herein by reference. Finally, BPI protein products are known to exhibit anti-mycobacterial activity, as described in co-owned, co-pending U.S. application Ser. No. 08/626,646 filed Apr. 1, 1996, which is in turn a continuation of U.S. application Ser. No. 08/285,803 filed Aug. 14, 1994, which is in turn a continuation-in-part of U.S. application Ser. No. 08/031,145 filed Mar. 12, 1993 and corresponding International Publication No. WO94/20129 (PCT/US94/02463), all of which are incorporated herein by reference.

[0018] The effects of BPI protein products in humans with endotoxin in circulation, including effects on TNF, IL-6 and endotoxin are described in co-owned, co-pending U.S. application Ser. No. 08/378,228, filed Jan. 24, 1995, which in turn is a continuation-in-part application of U.S. Ser. No. 08/291,112, filed Aug. 16, 1994, which in turn is a continuation-in-part application of U.S. Ser. No. 08/188,221, filed Jan. 24, 1994, and corresponding International Publication No. WO 95/19784 (PCT/US95/01151), all of which are incorporated herein by reference.

[0019] BPI protein products are also known to be useful for treatment of specific disease conditions, such as meningococcemia in humans (as described in co-owned, co-pending U.S. application Ser. No. 08/644,287 filed May 10, 1996, incorporated herein by reference), hemorrhagic trauma in humans, (as described in co-owned, co-pending U.S. application Ser. No. 08/652,292 filed May 23, 1996, incorporated herein by reference), burn injury (as described in U.S. Pat. No. 5,494,896 and corresponding International Publication No. WO 96/30037 (PCT/US96/02349), both of which are incorporated herein by reference), ischemia/reperfusion injury (as described in co-owned, co-pending U.S. application Ser. No. 08/232,527 filed Apr. 22, 1994, incorporated herein by reference), and liver resection (as described in co-owned, co-pending U.S. application Ser. No. 08/582,230 filed Jan. 3, 1996, which is in turn a continuation of U.S. application Ser. No. 08/318,357 filed Oct. 5, 1994, which is in turn a continuation-in-part of U.S. application Ser. No. 08/132,510 filed Oct. 5, 1993, and corresponding International Publication No. WO 95/10297 (PCT/US94/11404), all of which are incorporated herein by reference).

[0020] BPI protein products are also known to neutralize the anti-coagulant activity of exogenous heparin, as described in U.S. Pat. No. 5,348,942, incorporated herein by reference, as well as to be useful for treating chronic inflammatory diseases such as rheumatoid and reactive arthritis and for inhibiting angiogenesis and for treating angiogenesis-associated disorders including malignant tumors, ocular retinopathy and endometriosis, as described in co-owned, co-pending U.S. application Ser. No. 08/415,158 filed Mar. 31, 1995, which is in turn a continuation of U.S. application Ser. No. 08/093,202, filed Jul. 15, 1993, which is in turn a continuation-in-part of U.S. application Ser. No. 08/030,644, filed Mar. 12, 1993, all of which are incorporated herein by reference.

[0021] BPI protein products are also known for use in antithrombotic methods, as described in co-owned, co-pending U.S. application Ser. No. 08/644,290 filed May 10, 1996, incorporated herein by reference.

[0022] 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; biologically active polypeptide analogs of BPI protein or fragments or variants thereof, including cysteine-substituted analogs; and BPI-derived peptides. 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 incorporated herein by reference, discloses recombinant genes encoding, and methods for expression of, BPI proteins including recombinant BPI holoprotein, referred to as rBPI and recombinant fragments of BPI. U.S. Pat. No. 5,439,807 and corresponding International Publication No. WO 93/23540 (PCT/US93/04752), which are all incorporated herein 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.

[0023] Biologically active fragments of BPI (BPI fragments) include biologically active molecules that have the same or similar 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 1 to about 193 to 199 of natural human BPI, described in Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992), and referred to as rBPI23. In that publication, an expression vector was used as a source of DNA encoding a recombinant expression product (rBPI23) 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 figure 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: 145 and 146) set out in figure 1 of Gray et al., supra, with the exceptions noted for rBPI23 and with the exception that residue 417 is alanine (specified by GCT) rather than valine (specified by GTT). Other examples include dimeric forms of BPI fragments, as described in U.S. Pat. No. 5,447,913 and corresponding International Publication No. WO 95/24209 (PCT/US95/03125), all of which are incorporated herein by reference.

[0024] Biologically active variants of BPI (BPI variants) include but are not limited to recombinant hybrid fusion proteins, comprising BPI holoprotein or 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 in co-owned, copending U.S. application Ser. No. 07/885,911 filed May 19, 1992, and a continuation-in-part application thereof, U.S. application Ser. No. 08/064,693 filed May 19, 1993 and corresponding International Publication No. WO 93/23434 (PCT/US93/04754), which are all incorporated herein by reference 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.

[0025] Biologically active analogs of BPI (BPI analogs) include but are not limited to BPI protein products wherein one or more amino acid residues have been replaced by a different amino acid. For example, U.S. Pat. No. 5,420,019 and corresponding International Publication No. WO 94/18323 (PCT/US94/01235), all of which are incorporated herein by reference, discloses polypeptide analogs of BPI and BPI fragments wherein a cysteine residue is replaced by a different amino acid. A stable 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 rBPI21&Dgr;cys or rBPI21. Production of this N-terminal analog of BPI, rBPI21, has been described in Horwitz et al., Protein Expression Purification, 8:28-40 (1996). Other examples include dimeric forms of BPI analogs; e.g. U.S. Pat. No. 5,447,913 and corresponding International Publication No. WO 95/24209 (PCT/US95/03125), all of which are incorporated herein by reference.

[0026] 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 International Publication No. WO 95/19372 (PCT/US94/10427), which corresponds to U.S. application Ser. No. 08/306,473, filed Sep. 15, 1994, and International Publication No. WO94/20532 (PCT/US94/02465), which corresponds to U.S. application Ser. No. 08/209,762, filed Mar. 11, 1994, which is a continuation-in-part of U.S. application Ser. No. 08/183,222, filed Jan. 14, 1994, which is a continuation-in-part of U.S. application Ser. No. 08/093,202 filed Jul. 15, 1993 (corresponding to International Publication No. WO 94/20128 (PCT/US94/02401)), which is a continuation-in-part of U.S. application Ser. No. 08/030,644 filed Mar. 12, 1993, the disclosures of all of which are incorporated herein by reference.

[0027] As used herein, an “N-terminal BPI protein product” as differentiated from a “BPI protein product” includes natural, synthetic, and recombinant biologically active N-terminal polypeptide fragments of BPI protein having a molecular weight of about 25 kd or less; biologically active polypeptide analogs of these N-terminal BPI fragments, including cysteine-substituted analogs; biologically active polypeptide variants comprising such N-terminal BPI fragments or analogs thereof, including hybrid fusion proteins and dimers; and peptides derived from or based on N-terminal BPI protein having a molecular weight of about 25 kd or less (BPI-derived peptides).

[0028] Presently preferred BPI protein products include recombinantly-produced N-terminal fragments of BPI, especially those having a molecular weight of approximately between 20 to 25 kD such as rBPI21 or rBPI23, or dimeric forms of these N-terminal fragments (e.g., rBPI42 dimer). Preferred N-terminal dimeric products include dimeric BPI protein products wherein the monomers are N-terminal BPI fragments having the N-terminal residues from about 1 to 175 to about 1 to 199 of BPI holoprotein. A particularly preferred N-terminal dimeric product is the dimeric form of the BPI fragment having N-terminal residues 1 through 193, designated rBPI42 dimer. Additionally, preferred N-terminal BPI protein products include rBPI and BPI-derived peptides.

[0029] The administration of N-terminal BPI protein products is preferably accomplished with a pharmaceutical composition comprising an N-terminal BPI protein product and a pharmaceutically acceptable diluent, adjuvant, or carrier. The N-terminal BPI protein product may be administered without or in conjunction with known surfactants, other chemotherapeutic agents or additional known anti-chlamydial agents. A stable pharmaceutical composition containing BPI protein products (e.g., rBPI23) 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 stable pharmaceutical composition containing BPI protein products (e.g., rBPI21) 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 U.S. Pat. No. 5,488,034 and corresponding International Publication No. WO 94/17819 (PCT/US94/01239), the disclosures of all of which are incorporated herein by reference. As described in U.S. application Ser. No. 08/586,133 filed Jan. 12, 1996, which is in turn a continuation-in-part of U.S. application Ser. No. 08/530,599 filed Sep. 19, 1995, which is in turn a continuation-in-part of U.S. application Ser. No. 08/372,104 filed Jan. 13, 1995, and corresponding International Publication No. WO96/21436 (PCT/US96/01095), all of which are incorporated herein by reference, other poloxamer formulations of BPI protein products with enhanced activity may be utilized.

[0030] Therapeutic compositions comprising N-terminal BPI protein product may be administered systemically or topically. Systemic routes of administration include oral, intravenous, intramuscular or subcutaneous injection (including into a depot for long-term release), intraocular and retrobulbar, intrathecal, intraperitoneal (e.g. by intraperitoneal lavage), intrapulmonary (using powdered drug, or an aerosolized or nebulized drug solution), or transdermal.

[0031] When given parenterally, N-terminal BPI protein product compositions are generally injected in doses ranging from 1 &mgr;g/kg to 100 mg/kg per day, preferably at doses ranging from 0.1 mg/kg to 20 mg/kg per day, more preferably at doses ranging from 1 to 20 mg/kg/day and most preferably at doses ranging from 2 to 10 mg/kg/day. The treatment may continue by continuous infusion or intermittent injection or infusion, at the same, reduced or increased dose per day for, e.g., 1 to 3 days, and additionally as determined by the treating physician. When administered intravenously, N-terminal BPI protein products are preferably administered by an initial brief infusion followed by a continuous infusion. The preferred intravenous regimen is a 1 to 20 mg/kg brief intravenous infusion of N-terminal BPI protein product followed by a continuous intravenous infusion at a dose of 1 to 20 mg/kg/day, continuing for up to one week. A particularly preferred intravenous dosing regimen is a 1 to 4 mg/kg initial brief intravenous infusion followed by a continuous intravenous infusion at a dose of 1 to 4 mg/kg/day, continuing for up to 72 hours.

[0032] Topical routes include administration in the form of salves, creams, jellies, ophthalmic drops or ointments (as described in co-owned, co-pending U.S. application Ser. Nos. 08/557,289 and 08/557,287, both filed Nov. 14, 1995), ear drops, suppositories, irrigation fluids (for, e.g., irrigation of wounds) or medicated shampoos. For example, for topical administration in drop form, about 10 to 200 &mgr;L of an N-terminal BPI protein product composition may be applied one or more times per day as determined by the treating physician.

[0033] Those skilled in the art can readily optimize effective dosages and administration regimens for therapeutic compositions comprising N-terminal BPI protein product, as determined by good medical practice and the clinical condition of the individual patient.

[0034] “Concurrent administration” as used herein includes administration of the agents together, or before or after each other. The BPI protein products and second agent(s) may be administered by different routes. For example, the BPI protein product may be administered intravenously while the second agent(s) is(are) administered intramuscularly, intravenously, subcutaneously, orally or intraperitoneally. Alternatively, the BPI protein product may be administered intraperitoneally while the second agent(s) is(are) administered intraperitoneally or intravenously, or the BPI protein product may be administered in an aerosolized or nebulized form while the second agent(s) are administered, e.g., intravenously. The BPI protein product and second agent(s) may be both administered intravenously. The BPI protein product and second agent(s) may be given sequentially in the same intravenous line, after an intermediate flush, or may be given in different intravenous lines. The BPI protein product and second agent(s) may be administered simultaneously or sequentially, as long as they are given in a manner sufficient to allow all agents to achieve effective concentrations at the site of infection.

[0035] Other aspects and advantages of the present invention will be understood upon consideration of the following illustrative examples. Example 1 addresses the determination of BPI reactivity in the sera of ANCA-positive patients.

EXAMPLE 1

Measurement of BPI Antibody Titers in Sera of ANCA-Positive Patients

[0036] The presence of anti-BPI antibodies was determined in sera from 112 patients with confirmed ANCA-associated vasculitides (Wegener's granulomatosis, microscopic polyangiitis and Chung-Strauss syndrome), focusing on “double-negative” sera. Results are set forth in Table 1 below. 1 TABLE 1 BPI-ANCA in ANCA-associated vasculitides according to the main target antigens Wegener's Microscopic Chung-Strauss granulomatosis polyangutis syndrome +/&Sgr; % +/&Sgr; % +/&Sgr; % IIF/ANCA(+) 34/45 78 13/22 59 30/45 67 PR3(+) 18/35 51*  0/13 0  3/30 10 MPO(+)  0/35  0  8/13 62  5/30 17 BPI(+) 3†/35  9 1‡/13 8  0/30 0 Unknown 14/35 40  4/13 31 22/30 73 IIF/ANCA(−) 10/45 22  9/22 41 15/45 33 BPI(+)  0/10  0  0/9 0  3/15 20 * Low due to selection method. †No proteinase 3 (PR3) specificity. ‡No myeloperoxidase (MPO) specificity.

[0037] A considerable amount of sera that was ANCA negative by IIF proved to be BPI-reactive by ELISA, suggesting that screening for BPI reactivity by EUSA provides increased sensitivity compared to conventional indirect immunofluorescence techniques.

[0038] The sera of 102 patients with inflammatory bowel disease and primary sclerosing cholangitis were also evaluated for reactivity to BPI by ELISA. For comparison, 182 disease controls and 140 healthy blood donors were screened by means by ELISA using recombinant BPI (rBPI) as antigen (rBPI-ANCA-ELISA). Western blotting techniques were used to confirm the antigen recognized by these BPI-ANCA.

[0039] BPI-ANCA were detected in 36% of primary sclerosing cholangitis patients (44% of ANCA+patients), 26% of ulcerative colitis patients (29% of ANCA+) and 21% of Crohn's disease patients (24% of ANCA+), compared to less than 10% of ANCA-associated vasculitides (AAV) patients and disease controls.

[0040] In the group of AAV patients (selected according to ANCA status), 32% (36/112) were cANCA positive, 33.1% (37/112) pANCA positive, and 35% (39/112) were ANCA negative (table 1). In Wegener's granulomatosis, only 7% (3/45), in microscopic polyangiitis only 5% (1/22), and in Chung-Strauss syndrome only 7% (3/45) of sera were BPI-ANCA positive. All 3 BPI-ANCA-positive Chung-Strauss syndrome patients belonged to the ANCA negative subset.

[0041] In inflammatory bowel disease and primary sclerosing cholangitis, 52% (53/102) of patients were pANCA and 12.8% (13/102 were aANCA positive, while 35.2% (36/102) were ANCA negative (table 2). BPI-ANCA were present in 33% of ANCA-positive (by IIF) inflammatory bowel disease and primary sclerosing cholangitis patients. Primary sclerosing cholangitis showed the highest proportions of BPI-ANCA positively (44%), followed by ulcerative colitis (29%) and Crohn's disease (24%). In contrast to AAV, where BPI-ANCA do not further specify ANCA-positive and ANCA-negative sera, BPI-ANCA in inflammatory bowel disease and primary sclerosing cholangitis were also detected in 18% (primary sclerosing cholangitis, Crohn's disease) and 14% (ulcerative colitis) of “ANCA-negative” patients as assessed by IIF.

[0042] In the disease controls, none of the patients were cANCA positive, 31% (56/182) were pANCA and 10% (18/182) aANCA positive, while 59% (107/182) were ANCA negative. The sera from these patients did not show BPI-ANCA in significant numbers, either in the non-ANCA-associated vasculitis group (represented by ‘classic’ polyarteritis nodosa, Kawasaki's disease, Takayasu's and temporal arteritis, and Henoch-Schoeniein purpura) or in collagen vascular diseases (systemic lupus erythematosus [SLE], systemic sclerosis, polymyositis) and rheumatoid arthritis.

[0043] All 140 healthy blood donors were ANCA negative. Only one healthy donor was positive for BPI-ANCA as demonstrated by ELISA, exhibited no apparent clinical disease.

[0044] The frequency of BPI-ANCA in the diseases studied is shown according to their ANCA status (by IIF) in Table 2 below. 2 TABLE 2 BPI-ANCA+* (Total) (ANCA+)† (ANCA−)† Diseases n/&Sgr; (%) n/&Sgr; (%) n/&Sgr; (%) ANCA-associated vasculirides Wegener's granulomatosis  3/45     7% >10%‡ Chung-Strauss syndrome  3/45     7% Microscopic polyangiitis  1/22     5% Chronic inflammatoty bowel disease Crohn's Disease 10/44    23%  5/20 25% 5/24 21% Ulcerative colitis 20/54    37% 15/39 38% 5/15 33% Primary sclerosing 13/36    36% 11/25 44% 2/11 18% cholangitis Disease controls “Classical” polyarteritis  0/14     0% nodosa Kawasaki's Disease  0/9     0% Takayasu's Disease 1/10    10% <10%‡ Henoch-Schöenleim purpura  1/16     6% Temporal arteritis  4/42     9% Systemic lupus  2/28     7% <7%‡ erythematosus Systemic sclerosis  0/13    ≦0% Polymyositis  0/12     0% Rheumatoid arthritis  3/38     8% <10%† Healthy controls  1/140 <0.1% <0.1%‡ *ELISA. †Indirect immunofluorescence test screening. ‡Considered not significant.

[0045] In inflammatory bowel diseases (33.8%) and autoimmune liver disease (42.9%), anti-BPI antibodies are of major importance, appearing in more than one third of ANCA positive patients. BPI can thus be used as a recognized seromarker for a distinct disease entity.

[0046] To narrow the reactivity of the anti-BPI antibodies to a particular portion of the BPI molecule, all 60 of the samples that were reactive with BPI holoprotein (as shown in Table 2) were also screened with an N-terminal BPI protein product, rBPI21. Of these 60 samples, only 6 (12%) appeared to be reactive with rBPI21. A large amount of nonspecific binding was observed generally when rBPI21 was used as the antigen. This low level of reactivity with N-terminal BPI protein product indicated that the ANCA-recognized epitopes appear to reside predominantly outside the N-terminal 193 amino acids of BPI.

[0047] It has also been determined that BPI-reactive antibodies in cystic fibrosis patients are not reactive with the N-terminal BPI protein product rBPI21. See co-owned, co-pending U.S. application Ser. No. ______ [Carroll, “Improved Therapeutic Uses of BPI Protein Products in Cystic Fibrosis Patients”, Attorney Docket No. 27129/33581], filed concurrently herewith, incorporated herein by reference.

[0048] Numerous modifications and variations of the above-described invention are expected to occur to those of skill in the art. Accordingly, only such limitations as appear in the appended claims should be placed thereon.

Claims

1. In a method of treating a subject with a bactericidal/permeability-increasing (BPI) protein product, the improvement comprising administering an N-terminal BPI protein product to a subject having non-N-terminal-BPI-reactive antibodies.

2. The method of claim 1 wherein the N-terminal BPI protein product is being administered to a human subject to ameliorate adverse effects associated with endotoxin in circulation.

3. The method of claim 1 wherein the N-terminal BPI protein product is being administered to a human subject to ameliorate adverse effects associated with meningococcemia.

4. The method of claim 1 wherein the N-terminal BPI protein product is being administered to a human subject to ameliorate adverse effects associated with hemorrhagic trauma.

5. The method of claim 1 wherein the N-terminal BPI protein product is being administered to a subject to ameliorate adverse effects associated with burn injury.

6. The method of claim 1 wherein the subject being treated is suffering from a gram-negative bacterial infection.

7. The method of claim 1 wherein the subject being treated is suffering from a gram-positive bacterial or mycoplasmal infection.

8. The method of claim 1 wherein the subject being treated is suffering from a fungal infection.

9. The method of claim 1 wherein the subject being treated is suffering from a protozoan infection.

10. The method of claim 1 wherein the subject being treated is suffering from a chlamydial infection.

11. The method of claim 1 wherein the subject being treated is suffering from a mycobacterial infection.

12. The method of claim 1 wherein the subject being treated is suffering from a chronic inflammatory disease.

13. The method of claim 1 wherein the N-terminal BPI protein product is being administered to a subject to inhibit angiogenesis.

14. The method of claim 1 wherein the N-terminal BPI protein product is being administered to a subject to promote fibrinolysis.

15. The method of claim 1 wherein the N-terminal BPI protein product is an amino-terminal fragment of BPI protein having a molecular weight of about 20 kD to 25 kD.

16. The method of claim 1 wherein the BPI protein product is rBPI23 or a dimeric form thereof.

17. The method of claim 1 wherein the BPI protein product is rBPI21.