AU2005201015B2 - Therapeutic uses of BPI protein products in BPI-deficient humans - Google Patents

Description

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AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name of Applicant/s: Actual Inventor/s: XOMA Technology Ltd.

Ofer Levy Address for Service is: SHELSTON IP Margaret Street SYDNEY NSW 2000 CCN: 3710000352 Telephone No: Facsimile No.

(02) 9777 1111 (02) 9241 4666 Attorney Code: SW Invention Title: THERAPEUTIC USES OF BPI DEFICIENT HUMANS PROTEIN PRODUCTS IN BPI- Details of Original Application No. 40679/00 dated 03 Apr 2000 The following statement is a full description of this invention, including the best method of performing it known to me/us:- File: 33428AUP01 500546932 1.DOC5844 M\ -la- THERAPEUTIC USES OF BPI PROTEIN PRODUCTS IN BPI-DEFICIENT HUMANS SThis application is an Australian Divisional Patent Application of AU 40679/00, claiming an earliest priority date from US 09/285,124, of 1 April 1999, incorporated herein by reference.

Field of the Invention CN The present invention relates generally to novel therapeutic uses of BPI protein Sproducts that involve treatment of BPI-deficient subjects.

Background of the Invention Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Newborns as a group are at increased risk for invasive bacterial infections and resulting sepsis. Although the majority of these infections in newborns, are caused by gram-positive organisms, a variable but significant percentage of bacterial infections (about 20-40%) are due to gram-negative bacteria, particularly E. coli, Haemophilus influenzae, Klebsiella spp., and Enterobacter spp. In fact, it is the gram-negative infections that are, in some studies, associated with the highest mortality rate, which can be as high as about 40%. [Beck-Sague, CM et al., Pediatr Infect Dis J 13:1110-116 (1994) and Stoll, BJ et al., JPediatr 129:63-71 (1996)] The mechanisms by which newborns are at increased risk for these bacterial infections are not currently understood. Although the neutrophil defense system is innate, there are indications that its function at birth is immature and suboptimal.

Previos investigations of the activity of newborn neutrophils have demonstrated impaired adherence, chemotaxis, and phagocytosis. [Wright WC Jr. et al. Pediatrics 56:579-584 (1975); Cairo MS, AJDC, 143:40-46 (1989); Schelonka RL et al. Sem.

Perinatol., 22:2-14 (1998)]. Impaired stimulus-induced adhesion and migration has been associated with decreased surface expression of L-selectin and the P 2 -integrin Mac-1.

[Dinauer, MC, in "Hematology of Infancy Childhood", 5 th ed., Nathan and Orkin, eds., Col I, pp 889-967 (1998)]. These findings may explain the difficulty in mobilizing neutrophils to sites of bacterial infection but do not explain the decreased phagocytic and bactericidal activity of the neutrophils of newborns.

Most studies of the microbicidal mechanism of newborn neutrophils have focused on the oxidative mechanism the phagocyte oxidase/MPO/hydroxyl radical system), with conflicting data indicating either increased or decreased capacity of this oxygen-dependent mechanism in newborns. [Dinauer, supra, and Ambruso et al., PedRes 18:1148-53 (1984).] Despite a growing literature on antibiotic proteins and peptides, little is known about the oxygen-independent microbicidal mechanisms of newborn neutrophils.

A slightly decreased content of specific (secondary) granules in the neutrophils of newborns has been documented, with an associated modest 2-fold) decrease in lysozyme and lactoferrin content relative to adult neutrophils. [Ambruso et al., supra.] However, the major elements of the oxygen-independent antimicrobial arsenal ofneutrophil primary granules, including BPI and the defensin peptides, have not been assessed in neonates. Qing et al., Infect. Immun., 64:4638-4642 (1996), compared the lipopolysaccharide (LPS) binding of newborn neutrophils to that of adult neutrophils and reported that the newborn neutrophils have lower levels of membrane-associated 55-57 kDa and 25 kDa proteins capable of binding LPS. Although the missing proteins were not identified, the size and binding properties of the 55-57 kDa protein appeared to be similar to those of bactericidal/permeability-increasing protein (BPI) and the surface LPS receptor CD14.

The rising tide of antibiotic resistance has placed renewed emphasis on the development of agents to treat bacterial infection and its sequelae. Moreover, improved technology has led to increased survival rates for extremely ill full-term as well as premature neonates, which represent a growing population at high risk for bacterial infection. Although the replacement of neutrophils by granulocyte transfusion in newborns with sepsis has apparently been beneficial in some studies [Cairo et al., Pediatrics 74: 887-92 (1984)] this potential therapy has been complicated by difficulty in obtaining histocompatible neutrophils and by transfusion reactions.

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. nucleic acid and amino acid sequences are set out in SEQ ID NOS: 1 and 2 hereto. U.S. Patent Nos. 5,198,541 and 5,641,874 discloses recombinant genes encoding and methods for expression of BPI proteins, including BPI holoprotein and fragments of BPI. Recombinant human BPI holoprotein has also been produced in which valine at position 151 is specified by GTG rather than GTC, residue 185 is glutamic acid (specified by GAG) rather than lysine (specified by AAG) and residue 417 is alanine (specified by GCT) rather than valine (specified by GTT).

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 some endotoxin-neutralizing activity and only slightly detectable antibacterial 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 "rBPI, 3 has been produced by recombinant means and also retains antibacterial activity against gram-negative organisms. [Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992).] An N-terminal analog of BPI, designated rBPI 21 [rBPI(1-193)ala 1 32 has been produced as described in U.S. Patent No.

5,420,019 and Horwitz et al., Protein Expression Purification, 8:28-40 (1996).

An additional N-terminal analog of BPI, designated rBPI(10-193)C132A or rBPI(10-193)ala 1 32 has been produced as described in U.S. patent No. 6,013,631.

The bactericidal effect of BPI was originally reported to be highly specific to gram-negative species, 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, 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.

BPI protein products have a wide variety of beneficial activities.

BPI protein products are bactericidal for gram-negative bacteria, as described in U.S. Patent Nos. 5,198,541, 5,641,874, 5,948,408, 5,980,897 and 5,523,288.

International Publication No. WO 94/20130 proposes methods for treating subjects suffering from an infection gastrointestinal) with a species from the gram-negative bacterial genus Helicobacter with BPI protein products. BPI protein products also enhance the effectiveness of antibiotic therapy in gram-negative bacterial infections, as described in U.S. Patent Nos. 5,948,408, 5,980,897 and 5,523,288 and International Publication Nos. WO 89/01486 (PCT/US99/02700) and WO 95/08344 (PCT/US94/11255). BPI protein products are also bacterial for gram-positive bacteria and mycoplasma, and enhance the effectiveness of antibiotics in gram-positive bacterial infections, as described in U.S. Patent Nos.

5,578,572 and 5,783,561 and International Publication No. WO 95/19179 (PCT/US95/00498), and further as described for BPI-derived peptides in U.S. Patent No.

5,858,974, which is in turn a continuation-in-part of U.S. Application Serial No.

08/504,841 and corresponding International Publication Nos. WO 96/08509 (PCT/US95/09262) and WO 97/04008 (PCT/US96/03845), as well as in U.S. Patent Nos. 5,733,872, 5,763,567, 5,652,332, 5,856,438 and corresponding International Publication Nos. WO 94/20532 (PCT/US/94/02465) and WO 95/19372 (PCT/US94/10427). BPI protein products exhibit anti-protozoan activity, as described in U.S. Patent Nos. 5,646,114 and 6,013,629 and International Publication No. WO 96/01647 (PCT/US95/08624). BPI protein exhibit anti-chlamydial activity, as described in co-owned U.S. Patent No. 5,888,973 and WO 98/06415 (PCT/US97/13810). Finally, BPI protein products exhibit anti-mycobacterial activity, as described in co-owned, copending U.S. Application Serial No. 08/626,646, which is in turn a continuation-in-part of U.S. Application Serial No. 08/285,803, which in turn is a continuation-in-part of U.S. Application Serial No. 08/031,145 and corresponding International Publication No.

WO 94/20129 (PCT/US94/02463).

The effects of BPI protein products in humans with endotoxin are described in U.S. Patent Nos. 5,643,875, 5, 753,620 and 5,952,302 and corresponding International Publication No. WO 95/19784 (PCT/US95/01151).

BPI protein products are also useful for treatment of specific disease conditions, such as meningococcemia in humans (as described in U.S. Patent Nos. 5,888,977 and 5,990,086 and International Publication No. W097/42966 (PCT/US97/08016), hemorrhage due to trauma in humans, (as described in U.S. Patent Nos. 5,756,464 and 5,945,399, U.S. Application Serial No. 08/862,785 and corresponding International Publication No. WO 97/44056 (PCT/US97/08941), bur injury (as described in U.S.

Patent No. 5,494,896 and corresponding International Publication No. WO 96/30037 (PCT/US96/02349)) ischemia/reperfusion injury (as described in U.S. Patent No.

5,578,568), and depressed RES/liver resection (as described in co-owned, co-pending U.S. Application Serial No. 08/582,230 which is in turn a continuation of U.S.

Application Serial No. 08/318,357, which is in turn a continuation-in-part of U.S.

Application Serial 08/132,510, and corresponding International Publication No. WO 95/10297 (PCT/US94/11404).

BPI protein products also neutralize the anticoagulent activity of exogenous heparin, as described in U.S. Patent No. 5,854,214, and are useful for treating chronic inflammatory diseases such as rheumatoid and reactive arthritis, for inhibiting endothelial cell proliferation, and for inhibiting angiogenesis and for treating angiogeneis-associated disorders including malignant tumours, ocular retinopathy and endometriosis, as described in U.S. Patent Nos. 5,639,727, 5,807,818 and 5,837,678 and International Publication No. WO 94/20128 (PCT/US94/02401).

BPI protein products are also useful in antithrombotic methods, as described in U.S. Patent Nos. 5,741,779 and 5,935,930 and corresponding International Publication No. WO 97/42967 (PCT/US7/08017).

Summary of the Invention The present invention relates to novel therapeutic uses for BPI protein products that involve treatment of subjects, including humans, with a BPI deficiency condition, including a selective BPI deficiency. Another aspect of the invention provides treatment of newborns, including BPI-deficient newborns, with BPI protein products. The invention is based on the discovery that the neutrophils of newborns are selectivel deficient in BPI, a protein that plays an important role in defending against infection, including gram-negative bacterial infection. Treatment of subjects with a BPI deficiency condition is expected to alleviate adverse effects associated with this BPI deficiency, including, for example, decreasing susceptibility to infections, reducing the severity or invasiveness of the infection(s), and preventing the sequelae of the infection(s).

It is contemplated that the administration of a BPI protein product to a subject may be accompanied by the concurrent administration of other known therapeutic agents appropriate for treating the subject.

Use of a BPI protein product in the manufacture of a medicament for the treatment of humans with a BPI deficiency condition, including selective BPI deficiency condition, including selective BPI deficiency, or a medicament for the treatment of newborns, including BPI-deficient newborns, is also contemplated.

-7- 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.

According to the first aspect there is provided a method of treating a subject with BPI deficiency condition comprising administering to said subject an amount of BPI protein product effective to alleviate the BPI deficiency.

According to the second aspect there is provided a method of treating a subject with a BPI deficiency condition comprising administering to said subject an amount of BPI protein product, in combination with another therapeutic agent, effective to alleviate the BPI deficiency.

According to the third aspect there is provided a use of a BPI protein product in the manufacture of a medicament for the treatment and/or prevention of a BPI deficiency condition.

According to the fourth aspect there is provided a use ofa BPI protein product in combination with another therapeutic agent for the manufacture of a medicament for treating a subject with a BPI deficiency condition.

Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

Brief Description of the Drawings Figure 1 displays the relative BPI content of neonatal and adult neutrophils.

Detailed Description of the Invention The present invention provides novel therapeutic uses for BPI protein products that involve treatment of subjects with a BPI deficiency condition, including a selective BPI deficiency. Another aspect of the invention provides treatment of newborns, including BPI-deficient newborns, with BPI protein products. "Treatment" as used herein encompasses both prophylactic and therapeutic treatment.

The invention is based on the discovery that neonatal neutrophils are selectively deficient in BPI. On average, the neonatal neutrophils contained about 3fold less BPI than the adult neutrophils, yet both groups contained nearly identical levels of other microbicidal proteins MPO and defensin peptides) that are derived from the same primary (azurophil) granule compartment as BPI.

Although the average BPI content of newborn neutrophils was significantly lower than those of adults, it was not uniform. Some newborns apparently contain larger BPI stores than others. About 40% of newborns were markedly deficient to 10-fold less BPI than adults), with 33% of the neonatal samples showing no detectable levels of BPI at all. This variability suggests that BPI expression may be controlled by factors that are not uniformly distributed in newborns and may explain why some newborns are at greater risk of gramnegative bacterial infection than others.

The demonstration of such a BPI deficiency among newborns indicates that supplementation with BPI protein products may be of clinical benefit for newborns, including premature newborns. Newborns constitute a patient population that is at particularly high risk of infection and sepsis with subsequent poor outcomes. This demonstration of a BPI deficient condition, which has not previously been observed, also suggests that non-newborns, e.g., young children, older children or even adults, may also suffer from such a BPI deficiency and may benefit from supplementation with BPI protein products in amounts effective to alleviate the BPI deficiency. Such supplementation may provide a clinical benefit to such a BPI deficient subject. It is contemplated that supplementation is indicated whenever a BPI deficiency is observed or diagnosed, or whenever the subject is of a population with a high incidence of BPI deficiency newborns), even if the subject is not suffering from a condition associated with gram-negative bacteria and their endotoxin, for example, gram-negative bacterial infection, endotoxemia, or sepsis.

The invention thus contemplates methods for treating a subject with a BPI deficiency condition, including selective BPI deficiency, and methods for treating newborns, including BPI-deficient newborns, which comprises administering an amount of a BPI protein product effective to alleviate the adverse effects of BPI deficiency. The treatment of premature and full-term neonates, whether healthy or suffering from congenital defects, illnesses, or infections, is contemplated.

The BPI protein product also may provide an added advantage of enhancing the subject's resistance to or ability to fight infections, including gramnegative bacterial, gram-positive bacterial and fungal infections, and prevention of the sequelae thereof. The administration of BPI protein product is expected to reduce the incidence of severe or invasive infection and to also reduce the incidence of adverse sequelae of the infection. Such sequelae include, but are not limited to, a systemic inflammatory response, endotoxemia, bacterial and/or endotoxin-related shock and one or more conditions associated therewith, fever, tachycardia, tachypnea, cytokine overstimulation, increased vascular permeability, hypotension, complement activation, disseminated intravascular coagulation, anemia, thrombocytopenia, leukopenia, pulmonary edema, adult respiratory distress syndrome, intestinal ischemia, renal insufficiency and failure, and metabolic acidosis.

"BPI-deficient newborn" means that the newborn's neutrophils contain less BPI than the neutrophils of a normal adult. Correspondingly, a "BPI deficiency condition" means a condition in which the amount of BPI measured from the subject's neutrophils is less than the amount of BPI measured from the neutrophils of a normal adult. Although the exact level of BPI for comparison purposes to determine a "deficient" level will depend on the quantitation technique used, an exemplary standard value is approximately 230 ng per 106 neutrophils when a Western assay is used as described herein. Another exemplary standard value is 650 ng per 10 6 neutrophils when a radioimmunoassay is used as described in Weiss and Olson, Blood, 69:652-659 (1987).

A subject with "selective BPI deficiency" means that the subject's neutrophils contain less BPI than the neutrophils of a normal adult, yet have approximately normal levels of myeloperoxidase or defensins.

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 BPIderived 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.

Patent Nos. 5,198,541 and 5,641,874, the disclosures of which are incorporated herein by reference, disclose 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. Patent 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.

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, including those described in U.S. Patent Nos. 5,198,541 and 5,641,874. 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 rBPI 23 In that publication, an expression vector was used as a source of DNA encoding a recombinant expression product (rBPI2,) 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: 1 and 2) set out in Figure 1 of Gray et al., supra, with the exceptions noted for rBPI, 3 and with the exception that residue 417 is alanine (specified by GCT) rather than valine (specified by GTT). Another fragment consisting of residues 10-193 of BPI has been described in U.S. Patent No. 6,013,631, continuation-inpart U.S. Application Serial No. 09/336,402, filed June 18, 1999, and corresponding International Publication No. WO 99/66044 (PCT/US99/13860), all of which are incorporated herein by reference. Other examples include dimeric forms of BPI fragments, as described in U.S. Patent Nos. 5,447,913, 5,703,038, and 5,856,302 and corresponding International Publication No. WO 95/24209 (PCT/US95/03125), all of which are incorporated herein by reference.

Biologically active variants ofBPI (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 U.S. Patent No. 5,643,570 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.

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. Patent Nos.

5,420,019, 5,674,834 and 5,827,816 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 rBPIlAcys or.rBPI,,. Production of this N- -12terminal analog of BPI, rBPI 2 has been described in Horwitz et al., Protein Expression Purification. 8:28-40 (1996). Similarly, an analog consisting of residues 10-193 of BPI in which the cysteine at position 132 is replaced with an alanine (designated "rBPI(10-193)C132A" or "rBPI(10-193)ala 32 has been described in U.S. Patent No. 6,013,631, continuation-in-part U.S. Application Serial No. 09/336,402, filed June 18, 1999, and corresponding International Publication No. WO 99/66044 (PCT/US99/13860), all of which are incorporated herein by reference. Other examples include dimeric forms of BPI analogs; e.g.

U.S. Patent Nos. 5,447,913, 5,703,038, and 5,856,302 and corresponding International Publication No. WO 95/24209 (PCT/US95/03125), all of which are incorporated herein by reference.

Other BPI protein products useful according to the methods of the invention are peptides derived from or based on BPI produced by synthetic or recombinant means (BPI-derived peptides), such as those described in International Publication No. WO 97/04008 (PCT/US96/03845), which corresponds to U.S. Application Serial No. 08/621,259 filed March 21, 1996, and International Publication No. WO 96/08509 (PCT/US95/09262), which corresponds to U.S. Patent No. 5,858,974, and International Publication No. WO 95/19372 (PCT/US94/10427), which corresponds to U.S. Patent Nos. 5,652,332 and 5,856,438, and International Publication No. W094/20532 (PCT/US94/02465), which corresponds to U.S. Patent No. 5,763,567 which is a continuation of U.S. Patent No. 5,733,872, which is a continuation-in-part of U.S.

Application Serial No. 08/183,222, filed January 14, 1994, which is a continuation-in-part of U.S. Application Serial No. 08/093,202 filed July 1993 (corresponding to International Publication No. WO 94/20128 (PCT/US94/02401)), which is a continuation-in-part of U.S. Patent No.

5,348,942, as well as International Application No. PCT/US97/05287, which corresponds to U.S. Patent No. 5,851,802, the disclosures of all of which are incorporated herein by reference. Methods of recombinant peptide production are described in U.S. Patent No. 5,851,802 and International Publication No. WO 97/35009 (PCT/US97/05287), the disclosures of which are incorporated herein by reference.

Presently preferred BPI protein products include recombinantlyproduced N-terminal analogs and fragments of BPI, especially those having a molecular weight of approximately between 20 to 25 kD such as rBPI, or rBPI;,, rBPI(10-193)C132A (rBPI(10-193)ala'32), dimeric forms of these N-terminal proteins rBPI 42 dimer), and BPI-derived peptides.

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 or other therapeutic agents. A stable pharmaceutical composition containing BPI protein products rBPI 2 3 comprises the BPI protein product at a concentration of 1 mg/ml in citrate buffered saline (5 or 20 mM citrate, 150 mM NaC1, pH 5.0) comprising 0.1% by weight of poloxamer 188 (Pluronic F-68, BASF Wyandotte, Parsippany, NJ) and 0.002% by weight ofpolysorbate (Tween 80, ICI Americas Inc., Wilmington, DE). Another stable pharmaceutical composition containing BPI protein products rBPI 2 1 comprises the BPI protein product at a concentration of 2 mg/ml in 5 mM citrate, 150 mM NaC1, 0.2% poloxamer 188 and 0.002% polysorbate 80. Such preferred combinations are described in U.S. Patent Nos. 5,488,034, 5,696,090 and 5,955,427 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. Patent No. 5,912,228 and corresponding International Publication No.

W096/21436 (PCT/US96/01095), all of which are incorporated herein by reference, other poloxamer formulations of BPI protein products with enhanced activity may be utilized, optionally with EDTA.

Therapeutic compositions comprising 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 by intraperitoneal lavage), intrapulmonary (using powdered drug, or an -14aerosolised or nebulized drug solution), or transdermal.

When given parenterally, BPI protein product compositions are generally injected in doses ranging from 1 fig/kg to 100mg/kg per day, preferably at doses ranging from 0. 1 mg/kg to 20mg/kg per day, more preferably at doses ranging from 1 to 20 mg/kg/day and most preferably at doses ranging from 2 to 10mg/kg/day. The treatment may continue by continuous infusion or intermittent injection or infusion, at the same, reduced or increased dose per day for, 1 to 3 days, and additionally as determined by the treating physician. When administering intravenously, 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 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.

Topical routes include administration in the form of salves, creams, jellies, ophthalmic drops or ointments (as described in co-owned, U.S. Patent 5,686,414, filed November 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 pL of a BPI protein product composition may be applied one or more time per day as determined by the treating physician.

Those skilled in the art can readily optimise effective dosages and administration regimens for therapeutic compositions comprising BPI protein product, as determined by good medical practice and the clinical condition of the individual subject.

"Concurrent administration," or "co-administration," as used herein includes administration of the agents, in conjunction or combination, together, or before or after each other. The BPI protein product 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 intravenously, intramuscularly, subcutaneously, orally or intraperitoneally. The BPI protein product and second agent(s) may be given sequentially in the same intravenous line or may be given in different intravenous lines. Alternatively, the BPI protein product may be administered in a special form for gastric delivery, while the second agent(s) is(are) administered, e.g., orally. The formulated 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 action.

Other aspects and advantages of the present invention will be understood upon consideration of the following illustrative examples, which compare the components ofneutrophils from full term neonates and from adults.

Example 1 addresses the relative BPI content of neonatal and adult neutrophils.

Example 2 addresses the extracellular BPI levels of neonatal and adult blood.

Example 3 addresses the relative MPO and defensin peptide content of neonatal and adult neutrophils. Example 4 addresses effect of BPI protein product supplementation on antibacterial and cytokine-inducing activity of neonatal cord blood.

EXAMPLE 1 Comparison of BPI content of neonatal and adult neutrophils In order to compare the BPI content of neonatal and adult neutrophils, cell-associated BPI was measured by Western blot analysis of neutrophil detergent extracts. The neutrophil content of BPI was then estimated by visual comparison to two-fold dilutions of purified BPI, allowing quantitation of sample values.

Neonatal neutrophils were obtained from cord blood samples, which were collected immediately after cesarean section or vaginal delivery.

Cord blood was collected into sterile tubes anticoagulated with sodium heparin -16- (Becton Dickinson) and placed on ice. All samples were labeled numerically and the results kept anonymous. Adult neutrophils were obtained from peripheral blood from healthy adult volunteers.

Neutrophils were isolated from whole blood as described in Levy et al., J. Immunol 154: 5403-10 (1995). Anticoagulated blood was promptly (within 30-60 minutes) processed by dextran sedimentation pyrogen-free dextran (United States Biochemical) diluted in Hanks Balanced Salt Solution without divalent cations, to avoid neutrophil clumping (Gibco BRL)]. Ficollhypaque (endotoxin-free Ficoll-Paque Plus, Pharmacia Biotech) gradient centrifugation was employed to generate a neutrophil-rich fraction. Brief hypotonic lysis (-45 sec on ice) was employed to remove red blood cells. An automated total WBC count and differential (Technion H3 RTX automated cell counter, Miles) was obtained on every sample prior to pelleting by centrifugation.

White cell differential counts were often confirmed by Wright stain and manual assessment. Neutrophil viability was assessed by trypan blue exclusion.

Neutrophil pellets (typically >85% pure) were frozen in Eppendorf tubes at 0 C prior to batch analysis.

Western blots to determine relative BPI content were conducted as follows. Neutrophils were thawed and solubilized with 4X SDS-PAGE loading buffer SDS, 0.34 glycerol, 0.04% Bromphenol Blue, 0.02 M DTT, 0.2 Tris pH 6.8) prior to fractionation over a 10% SDS-PAGE gel (PAGE-ONE precast 10% gels, Owl Separation System). After Western transfer onto nitrocellulose (Protran BA85, pore size 0.45 kim, Schleicher Schuell), and blocking of non-specific sites with 3% bovine serum albumin [BSA/Tris-buffered saline pH 7.4 BPI was detected using 0.1% whole anti-BPI goat serum as described in Levy et al.,JClin Invest 94:672-682. (1994). Bound antibody was detected using: 0.05% peroxidase-conjugated protein G followed by metal-enhanced diaminobenzoic acid (DAB; Pierce), 1:35,000 dilution of peroxidase-conjugated protein G as part of the SuperSignal chemiluminescent system (Pierce), or 0.1% 1-125 protein G. For detection methods and signal was detected by exposing the blots to Kodak XAR film. This Western transfer protocol provided detection in the range of 200 nanograms with readily apparent differences in signal intensity between twofold dilutions of a BPI standard, thus allowing interpolation of BPI content in test samples. Recombinant human BPI (rBPI 50 was prepared as described in Horwitz et al., Protein Expression Purification 8: 28-40 (1996).

To compare analysis of BPI content by two independent techniques, a number ofneutrophil samples were extracted with sulfuric acid to solubilize BPI as described in Levy et al. (1994), supra, and analyzed for BPI content by both Western blotting and a sandwich ELISA assay as described in White et al., JImmunol Methods 167:227-235 (1994). Similar BPI levels were obtained by both techniques, indicating that the Western blotting data are representative and relatively accurate.

Composite data from multiple Western blotting experiments are shown in Figure 1, which is a scattergram of BPI content in the neutrophils of newborns and adults. Horizontal bars indicate average values for newborns and adults. All samples of adult neutrophils (n 22, mean age 29 years) contained quantifiable levels of BPI, the average of which was 234 27 ng per 10 6 neutrophils. In contrast, newborn neutrophils (n 21, mean gestational age 38.6 weeks) contained significantly lower amounts of BPI: 67 13 ng per 106 neutrophils p< 0.001, 2-sided test). Median values for BPI content of adult and newborn neutrophils were 200 ng and 50 ng respectively. Thus, newborn neutrophils contained at least 3-fold less BPI than adult neutrophils.

It is also evident from Figure 1 that about 40% (8 of 21) of the newborn neutrophils were markedly deficient in BPI. Among these eight samples, seven had no detectable BPI even after prolonged exposure. This number represents 33% of the newborn patients studied. For the purposes of quantitation, such samples were considered to contain one-half of the lowest amount of BPI that was detectable in the standard curve about 10 ng per 106 neutrophils). The analysis of BPI content was thus conservatively biased towards overestimating the amount of BPI in newborn neutrophils, with the actual difference in neutrophil BPI content of some newborns relative to adults perhaps -18being more than 10-fold. Of note, MPO was easily detected in three newborns in whom there was no detectable BPI.

EXAMPLE 2 Comparison of extracellular levels of BPI in neonatal and adult plasma To determine whether the relatively low BPI content of newborn neutrophils was related to degranulation, possibly secondary to perinatal stress, the levels of extracellular BPI in newborn plasma samples and adult plasma samples were compared. Newborn and adult plasma samples were collected within 30-60 minutes of drawing cord or peripheral venous blood, respectively.

Samples were stored in cryogenic microtubes (Sarstedt) at -70 0 C prior to batch analysis.

BPI content of plasma was determined employing a biotinylated anti-BPI antibody in a sandwich ELISA format as described in White et al.

(1994), supra. This ELISA system yielded a linear range from 0.1 to 6 ng BPI/ml and showed negligible cross reactivity with the homologous lipopolysaccharidebinding protein (LBP).

The average cord plasma BPI content was 16 ng/ml (n 13), which is higher than that previously reported for plasma samples collected from 20 healthy adults to 2.1 ng/ml; White et al. (1994), supra). However, calculated per cc of whole cord blood, this plasma content of BPI represents less than 2% of cellular BPI content. Thus, there was no evidence for substantial extracellular degranulation of BPI at the time immediately preceding collection and processing of newborn cord blood.

EXAMPLE 3 Comparison of MPO and defensin levels in neonatal and adult neutrophils To assess whether other primary (azurophil) granule constituents were also relatively decreased in newborn neutrophils, the content of myeloperoxidase (MPO) and of defensin peptides in neonatal and adult neutrophils was measured as follows.

Levels of myeloperoxidase (MPO) were detected by Western blotting using 0.1% rabbit anti-MPO serum [described in Nauseef et al., J Clin Invest 71: 1297-1307 (1983)] followed by 0.1% 1' 25 protein G. As control for MPO blots, a two-fold dose curve of adult azurophil granule fraction (prepared as described in Borregaard et al., J Cell Biol 97: 52-61 (1983) was solubilized in 4X SDS-PAGE buffer and analyzed as well. For purposes of quantitation, MPO content in neutrophil samples was expressed in "antigenic units" defined in relation to an adult azurophil granule extract standard: one antigenic unit was set equal to the band intensity of an azurophil granule extract sample representing 10 6 adult neutrophil equivalents.

Levels of defensins were detected by subjecting neutrophil extracts from adults and newborns to acid-urea (AU)-PAGE as described in Harwig et al., Meth Enzymol 236: 160-172 (1994). Briefly, neutrophils were sonicated in acetic acid prior to overnight extraction at 4°C. Insoluble components were removed by centrifugation, supernatants lyophilized, and resuspended in AU- PAGE buffer prior to electrophoresis and Coomassie Brilliant Blue R stain. For each samples, the intensity of staining was visually compared to two-fold dilutions of control extracts.

The results of Western blotting for MPO showed that the MPO content of newborn neutrophils (6.0 2.5 antigenic units per 106 cells, n=7 samples) and of adult neutrophils (4.3 1.6 antigenic units per 10 6 cells, n=7 samples) was not statistically different. Thus, in accordance with previous observations by others (Kjeldsen et al., Pediatr Res 40: 120-129 (1996), newborn and adult neutrophils appear to contain nearly identical amounts of MPO.

Despite the use of a sensitive detection technique which easily revealed two-fold differences in defensin content, the AU-PAGE results for the defensin peptides showed that there was no discernible difference in the content ofdefensins in adult and newborn neutrophils. Of note, although the levels of lysozyme were somewhat decreased in some of the newborn neutrophil samples, the overall pattern ofneutrophil proteins did not significantly vary in migration or band intensity between newborn and adults.

Taken together, the results in Examples 1, 2 and 3 indicate that newborn neutrophils have intrinsically lesser quantities of BPI because: a priori considerations would predict that BPI should remain intracellularly since it resides in the primary (azurophilic) granules which are known, to be the least easily mobilized compartment of both adult and newborn neutrophils, (b) newborn and adult neutrophils contain nearly identical amounts of both MPO and defensin, both of which are components of the same primary (azurophil) granule where BPI is stored, and it is highly unlikely that selective degranulation of BPI occurred, and levels of BPI in cord plasma represent only a small fraction 2 of total cellular BPI, suggesting that there was no significant release of BPI from cellular stores to the extracellular space at the time immediately preceding cord blood collection.

EXAMPLE 4 Effect of BPI protein product supplementation on antibacterial activity of newborn cord blood This experiment evaluated the effect of supplementation of exogenous BPI protein product to newborn cord blood, as measured by effect on survival and TNF-a cytokine-inducing activity of various gram-negative bacteria.

Cord blood samples were collected immediately after vaginal delivery (n=l 7) or cesarean section (n=26) into sterile tubes anticoagulated with sodium citrate [sodium citrate (0.129M, tubes, Becton Dickinson (Franklin Lakes, NJ)].

The bacteria tested were E. coli Kl/r, a Kl-encapsulated, rough LPS, serum-resistant clinical isolate which has been shown to be sensitive to BPI- -21mediated killing both in artificial media and whole adult blood ex vivo; Citrobacter koseri, isolated from the blood and cerebrospinal fluid of a 14 day old male with meningitis; and Klebsiella pneumoniae, Enterobacter agglomerans and Enterobacter cloaceae, as well as Serratia marcescens isolated from blood cultures of newborns (7-27 day old) with congenital cardiac defects requiring invasive surgery. Frozen stocks of bacteria were prepared by culturing in trypticase soy broth [TSB, Becton Dickinson Co., Cockeysville, MD], and adding sterile glycerol to 15% (vol/vol) prior to freezing at 80 0

C.

For bactericidal assays, subcultures of bacterial stocks were prepared by inoculating a loopful into 20 ml of TSB and incubating at 37 C with shaking for about 4 hours (to late logarithmic phase growth). Bacterial concentration was determined by measuring optical density (OD) at 550 nm in a spectrophotometer. Subcultures were harvested by centrifugation and resuspended in sterile physiologic saline to the desired concentration.

Antibacterial assays were conducted in Eppendorf tubes [Research Products International (Mount Prospect, IL)] in a total volume of 100 1l. Samples contained 80 ,l cord blood or buffered saline (20 mM sodium phosphate pH 7.4/0.9% NaCI) as a control, 10 M1 rBPI 2 1 (or buffered saline), and 10 A1 of bacteria (added last, to a final concentration of about 10 4 Samples were incubated with shaking at 37 C. At 0, 30, 90, and 180 minutes, 10 l of each sample was plated on a Petri dish and dispersed with about 9 mL of molten Bactoagar containing 0.8% (wt/vol) nutrient broth [Difco Laboratories (Detroit, MI)] and 0.5% (wt/vol) NaC1. The agar was allowed to solidify at room temperature, and bacterial viability was measured as the number of colonies formed after incubation of plates at 37 0 C for 18 to 24 hours. Bacterial viability was expressed as colony forming units (CFU) as a percentage of the buffered saline control sample.

For cytokine-induction assays, bacteria were incubated in blood for 5 hours to allow accumulation of TNF-a. Blood was diluted five-fold with RPMI [Gibco BRL, Grand Island, NY] then centrifuged at 1000 x g for 5 min to collect the extracellular fluid (diluted plasma). Samples were stored frozen at -22- 0 C prior to measurement of TNF-a using a Quantikine TNF-a ELISA Kit [R&D System (Minneapolis, MN)] according to the manufacturer's instructions.

Results showed that while growth ofE. coli Kl/r was inhibited by adult blood, newborn cord blood served as a growth medium for this organism which grew logarithmically over several hours. Addition ofrBPI 2 1 was able to markedly diminish growth of this organism both in adult and newborn cord blood with an IC50 of about 10 nM. Similarly, growth of Citrobacter koserii was inhibited by adult blood but the organism grew logarithmically in newborn cord blood. Addition ofrBPI 2 provided a reduction in bacterial growth with an ICso of about 1000 nM. K. pneumoniae, E. cloaceae and S. marcescens were relatively resistant to BPI protein product, while E. agglomerans was too rapidly killed by both adult and newborn cord to observe an effect of BPI protein product.

Cytokine responses of newborn cord blood to E. coli Kl/r were similar to those of adult blood. Addition of rBPI, was able to inhibit bacteriainduced TNF-a release with similar potency in both adult and newborn cord blood (ICso about 10-100 nM). C. koserii, Kpneumoniae, E. cloaceae and agglomerans, and S. marcescens also induced substantial TNF-a release in both adult and newborn cord blood. The overall average TNF-a release induced by all of the six gram-negative isolates tested was closely similar in both adult and newborn cord blood. Addition of rBPI 2 was able to inhibit induction of TNF-a release by all of the species tested (IC,5 ranging from about 1 to 1000 nM; C.

koserii about 100 nM, K. pneumoniae about 10-100 nM, E. cloaceae about 100 nM, E. agglomerans about 10-100 nM and S. marcescens about 1-10 nM).

Thus, these results demonstrated that replenishment of BPI in the form of rBPI 2 1 enhanced the antibacterial activity of newborn cord blood against E. coli K1/r and C. koserii and inhibited bacteria-induced cytokine release.

-23- 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 (28)

1. A method of treating a subject with a BPI deficiency condition comprising administering to said subject an amount of BPI protein product effective to alleviate the BPI deficiency.

2. A method of treating a subject with a BPI deficiency condition comprising administering to said subject an amount of BPI protein product, in combination with another therapeutic agent, effective to alleviate the BPI deficiency.

3. A method according to claim 2, wherein said another therapeutic agent is administered concurrently.

4. A method according to any one of claims 1 to 3, wherein the treatment is for decreasing susceptibility to infection, or for reducing the severity or invasiveness of infection. A method according to any one of claims 1 to 4, wherein the treatment is for preventing or for reducing the incidence of adverse sequelae of infection, including a systemic inflammatory response, endotoxemia, bacterial and/or endotoxin-related shock, fever, tachycardia, tachypnea, cytokine overstimulation, increased vascular permeability, hypotension, complement activation, disseminated intravascular coagulation, anemia, thrombocytopenia, leukopenia, pulmonary edema, adult respiratory distress syndrome, intestinal ischemia, renal insufficiency and failure, or metabolic acidosis.

6. A method according to claim 4 or 5, wherein the infection is gram-negative bacterial infection, gram-positive bacterial infection or fungal infection.

7. A method according to any one of claims 1 to 6, wherein the treatment is systemic or topical.

8. A method according to claim 7, wherein the administration is parenteral at a dose ranging from 1 jig/kg to 100 mg/kg per day.

9. The method according to any one of claims 1 to 8, wherein the subject is an adult. The method according to any one of claims 1 to 9, wherein the condition is a selective BPI deficiency.

11. The method according to any one of claims 1 to 8, wherein the subject is a newborn.

12. The method according to claim 11 wherein the newborn is a premature newborn.

13. The method according to any one of claims 1 to 12, wherein the BPI protein product is rBPI 21

14. The method according to any one of claims 1 to 12, wherein the BPI protein product is an N-terminal fragment of BPI having a molecular weight approximately between 20 to 25 kd. The method according to any one of claims 1 to 12, wherein the BPI protein product is rBPI(10-193)ala 132

16. The method according to any one of claims I to 12, wherein the protein product is rBP 50

17. Use of a BPI protein product for the manufacture of a medicament for treating a subject with a BPI deficiency condition.

18. Use of a BPI protein product in combination with another therapeutic agent for the manufacture of a medicament for treating a subject with a BPI deficiency condition.

19. Use according to claim 17 for concurrent administration with another therapeutic agent. A use according to any one of claims 17 to 19, wherein the medicament is for decreasing susceptibility to infection, or for reducing the severity or invasiveness of infection.

21. A use according to any one of claims 17 to 20, wherein the medicament is for preventing or for reducing the incidence of adverse sequelae of infection, including a systemic inflammatory response, endotoxemia, bacterial and/or -26- endotoxin-related shock, fever, tachycardia, tachypnea, cytokine overstimulation, increased vascular permeability, hypotension, complement activation, disseminated intravascular coagulation, anemia, thrombocytopenia, leukopenia, pulmonary edema, adult respiratory distress syndrome, intestinal ischemia, renal insufficiency and failure, or metabolic acidosis.

22. A use according to claim 20 or 21, wherein the infection is gram-negative bacterial infection, gram-positive bacterial infection or fungal infection.

23. A use according to any one of claims 17 to 22, wherein the medicament is for systemic or topical administration.

24. A use according to claim 23, wherein the medicament is for parenteral administration in a dose ranging from 1 ptg/kg to 100 mg/kg per day. Use according to any one of claims 17 to 24, wherein the subject is an adult.

26. Use according to any one of claims 17 to 24, wherein the condition is a selective BPI deficiency.

27. Use according to any one of claims 17 to 24, wherein the subject is a newborn.

28. Use according to claim 27 wherein the newborn is a premature newborn.

29. Use according to any one of claims 17 to 28, wherein the BPI protein product is rBPI 21 Use according to any one of claims 17 to 28, wherein the BPI protein product is an N-terminal fragment at BPI having a molecular weight approximately between 20 to 25 kd.

31. Use according to any one of claims 17 to 28, wherein the BPI protein product is rBPI(10-193)ala 1 32

32. Use according to any one of claims 17 to 28, wherein the BPI protein product is rBPI 50 \O -27-

33. A method of treating a subject with a BPI deficiency condition substantially as z herein described with reference to any one of the embodiments of the invention illustrated in the accompanying examples.

34. Use of BPI protein product in the manufacture of a medicament substantially as tt 5 herein described with reference to any one of the embodiments of the invention _illustrated in the accompanying examples. Dated this 21st day of November 2006 Shelston IP Attorneys for: XOMA Technology Ltd.