FIELD OF THE INVENTION The present invention relates generally to novel therapeutic uses of BPI protein products that involve inhibition of adenosine triphosphatase (ATPase) activity in mammals.

BACKGROUND OF THE INVENTION H+/K--ATPases are present in cytoplasmic membranes of eukaryotic cells and act as proton pumps. In particular, an H+/K+-ATPase is present in the resting gastric parietal cell membrane and mainly in the secretory canaliculus in the stimulated parietal cell. A small quantity of an H+/K~-ATPase is also expressed in kidneys. In the parietal cell. its function is to secrete acid by forcing an exchange of H30-for K-. In the kidneys it is probably responsible for a small part of the elimination of an acid load, whereas the homologous colonic H~/K~-ATPase is likely involved in potassium homeostasis. Gastric HT/K"- ATPase is a member of the large family of P-type ATPases, so called because the catalytic protein undergoes a cycle of phosphorylation and dephosphorylation as part of the transport process. However, its amino acid sequence and function are unique. [Sachs, PharrnacotlieraPv, 17 : 2 ?- ? 7 (1997).] Other members of the extended family of P-type ATPases include the yeast H/K-ATPase (which regulates intracellular pH, ion balance and nutrient uptake by the cell), the mammalian Na~/K~ ATPases (e g., in kidney) and

the mammalian Ca'T-ATPase (e. g., in muscle). Within the P-type ATPase class, about 25% of the primary structure is held in common, with structural conservation observed most strongly in a set of six sequences found in the cytoplasmic catalytic domains. These conserved primary sequence elements are located within the putative transduction and kinase domains, including the ATP binding site and the phosphorylation site. The overall pattern of secondary structural features, including the general location and number of transmembrane segments, is also similar among all the P-type ATPases. [Monk and Perlin, Crit.

Rew. Microbiol., 20: 209-223 1994).] The family of P-type ATPases shares little homology with the distinct family of Fo-F, ATPases, which includes the H*/K'- ATPases of bacteria, mitochondria and chloroplasts and the ATP synthases from mitochondria. [Serrano et al., Nature, 319: 689-693 (1986).] A number of disease states and conditions involve excess acid secretion. Because there are a number of pathways for stimulating acid secretion, drugs that act by inhibiting the last step of acid secretion are preferable to drugs that act earlier in the pathways. For example, omeprazole (a pyridinyl-2- methylenesulfinyl-2 benzimidazole derivative) acts by inhibiting the gastric parietal cell H-/Kw ATPase. This enzyme plays an important role in the actual secretion of HCI. Blockade of the pump inhibits acid secretion regardless of the pathway of stimulation that is being used by the cell.

The lifetime prevalence of peptic (gastric and duodenal) ulcer disease is approximately 10%, and some physicians estimate that 50% of healthy individuals experience heartburn on a daily basis. The causes of peptic ulcer disease and gastrointestinal inflammatory diseases are not fully understood, but they can be induced by stress, by gastric cancers, by drugs such as aspirin, non- steroidal anti-inflammatory drugs (NSAIDs), e. ., indomethacin, ibuprofen, naproxen, tolmetin, sulindac, piroxicam, diflunisal, fenoprofen, and possibly glucocorticoids, by ingestion of corrosive chemicals, such as strong acids or strong alkali, and by pathogenic viruses and microorganisms. Peptic ulcers are a part of Zollinger-Ellison syndrome, which is characterized bv gastric acid

hypersecretion caused by a gastrin-secreting tumor of the pancreatic islet cells.

Helicobacter pvlori infection is frequently present in patients with peptic ulcer disease and has been proposed as a contributory or modifying factor therefor. In addition, eosinophilic gastritis, granulomatous gastritis and prior gastnc surgery may produce gastritis or gastric ulceration. [Harrison's Principles of Internal Medicine, 13th ed., Isselbacher et al., eds., McGraw-Hill, NY (1994), pages 1363-1382.] Gastric acidity is an important factor in the development and exacerbation of these diseases, and agents that reduce gastric acidity effectively promote healing. Such drugs include Hr histamine receptor antagonists (e. g., cimetidine, ranitidine, famotidine, nizatidine) and covalent inhibitors of the H+/K~-ATPase of the parietal cell (e. g., omeprazole, lansoprazole). Protective agents such as sucralfate, colloidal bismuth, prostaglandin agonists (such as misoprostol or other prostaglandin E, and E, derivatives) and antacids are also effective. Outside of the United States, muscarinic cholinergic antagonists (e. g., prenzepine and telenzepine) and carbenoxolone have been used to treat peptic ulcer disease. [Goodman & Gilman, The Pharmacological Basis of Therapeutics, 9th ed., McGraw-Hill, NY (1996), pages 901-915.] Other noncovalent gastric H-/K--ATPase antagonists include the K~-competitive drugs SCH 28080 and SK&F96067. [Monk and Perlin, Crit. Rev. Microbiol., 20: 209-223 (1994).] However, there remains a need for agents which are H'/K-- ATPase inhibitors, capable of inhibiting gastric acid secretion and ameliorating the symptoms of peptic ulcer disease and gastrointestinal inflammatory diseases, including those not involving Helicobacter infection.

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. cllenZ 4 : 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 U. S. Patent No.

5,198,541 and Figure 1 of Gray et al., J. Biol. Chenu., 264: 9505 (1989), incorporated herein by reference. The Gray et al. nucleic acid and amino acid sequence are set out in SEQ ID NOS: 1 and 2 hereto. U. S. Patent No. 5,198,541 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), supr-a.] 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. Chenu.. 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"rBPI2R,"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 designated rBPI,, (also referred to as rBPI (1-193) ala'~~) has been described in U. S. Patent No.

5,420,019.

The bactericidal effect of BPI was originally reported to be highly specific to gram-negative species, e. (-,., in Elsbach and Weisss 1/7flU1171171atiO) I.

Basic Principles aad Clirtical Corwelates, 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.

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 (e. g. 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 bactericidal 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 and International Publication No. WO 95/19180 (PCT/US95/00656). BPI protein products exhibit antifungal activity, and enhance the activity of other antifungal agents, as described in U. S. Patent No. 5n627sl53 and International Publication No. WO 95/19179

(PCT US95/00498), and further as described for BPI-denved 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 products 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, co-pending U. S. Application Serial No. 08/626,646, which is in turn a continuation of U. S. Application Serial No. 08/285,803, which is in turn 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 in circulation, including effects on TNF, IL-6 and endotoxin are described in U. S.

Patent Nos. 753,620 and 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), burn 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 No. 08/132,510, and corresponding International Publication No. WO 95/10297 (PCT/US94/11404).

BPI protein products also neutralize the anticoagulant activity of exogenous heparin, as described in U. S. Patent No. 5,348,942, neutralize heparin iii vitro 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 angiogenesis-associated disorders including malignant tumors, 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 provides novel therapeutic uses for BPI protein products, including BPI-derived peptides, that involve inhibition of Ht/K- ATPase activity, including methods for inhibiting gastric acid secretion. Uses of BPI protein products according to the invention are specifically contemplated in mammals, particularly humans, for prophylactic or therapeutic treatment of disease states or conditions exacerbated by acid secretion involving H-/K- ATPase activity, such as gastrointestinal ulcer disease, gastrointestinal inflammatory diseases or other conditions exacerbated by gastric acidity, including, for example, gastroesophageal reflux disease (GERD), esophagitis, gastritis, duodenitis, gastric cancers, gastrinomas, Zollinger-Ellison syndrome. acute upper gastrointestinal bleeding, gastric ulcers, duodenal ulcers, ingestion of corrosive chemicals. stress ulcers, aspiration pneumonia. chronic or excessive

alcohol consumption, patients in intensive care situations, or pre-andior post- operatively to prevent aspiration of gastric acid.

One aspect of the invention provides a method of inhibiting H-/K- ATPase activity in a mammal in need thereof comprising administering to said mammal a therapeutically effective amount of a BPI protein product.

Another aspect of the invention provides a method of inhibiting gastric acid secretion in a mammal in need thereof comprising administering to said mammal a therapeutically effective amount of a BPI protein product.

Exemplary BPI protein products include recombinantly-produced N-terminal analogs or fragments of BPI, especially those having a molecular weight of approximately between 20 to 25 kD such as rBPI,,, rBPI23, rBPI (10- 193) C132A, (rBPI (10-193) ala'32), dimeric forms ofthese N-terminal polypeptides (e. g., rBPI42 dimer), or BPI-derived peptides. Exemplary BPI-derived peptides include XMP. 391 (SEQ ID NO: 4), XMP. 416 (SEQ ID NO: 5) or XMP. 445 (SEQ ID NO: 6) [the structure and activity of which are described in co-owned, co-pending U. S. Serial No. U. S. Serial No. 09/406,243 filed September 24,1999, incorporated herein by reference].

It is contemplated that the administration of a BPI protein product may be accompanied by the concurrent administration of other therapeutic agents, such as agents that also inhibit gastric acid secretion, protect the gastric mucosa, or neutralize gastric acids. It is also contemplated that a BPI protein product may be concurrently administered with agents that tend to induce gastric injury, such as aspirin, NSAIDs, glucocorticoids, or alcohol.

The invention also provides methods of screening BPI protein products, including BPI-derived peptides. for inhibition of H-/K-ATPase activity.

Such methods would comprise steps of, e. g., contacting an H'/K-ATPase with a BPI protein product and measuring H-/K-ATPase activity in the presence and absence of the BPI protein product. H-/K-ATPase activity can be measured directly, through ATP phosphatase/synthase assays, or it can be measured indirectly. e. g.. througll detectin (J acidification of medium or any one of the other

assays described herein. Optionally the screening methods involve a further step of testing candidates in animal models of gastrointestinal inflammatory conditions that are exacerbated by gastric acidity.

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.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 displays effects of BPI protein products on phosphatase activity of microsomal preparations.

Figure 2 displays effects of XMP. 416 (SEQ ID NO: 5) on pH and 3H-thymidine measurements in a culture of whole cells.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel therapeutic uses for BPI protein products, particularly BPI-derived peptides, that involve inhibition of H-/K-ATPase activity, or methods for inhibiting acid secretion, including acid secretion by gastric parietal cells, or methods for increasing potassium excretion.

The invention is based on the novel discovery that BPI protein products inhibit the H-/K'ATPase activity of plasma membranes and inhibit acid secretion by cells. BPI protein product are administered in amounts effective to inhibit such H/K ATPase activity or in amounts effective to inhibit acid secretion.

"Treatment"as used herein encompasses both prophylactic and/or therapeutic treatment.

Therapeutic uses of BPI protein products are specifically contemplated for treatment of mammals, including humans, suffering from gastrointestinal ulcer disease or gastrointestinal inflammatory diseases or other conditions exacerbated by gastric acidity, including, for example, gastroesophageal reflux disease (GERD) esophagitis, gastritis, duodenitis,

gastrinomas, Zollinger-Ellison syndrome, acute upper gastrointestinal bleeding, gastric ulcers, duodenal ulcers or stress ulcers, including gastritis or ulcer conditions not involving persistent Helicobacter infection. Furthermore, BPI protein product treatment is contemplated for patients being treated with drugs that induce gastric injury, such as aspirin, non-steroidal anti-inflammatory drugs (NSAIDs), e. g., indomethacin, ibuprofen, naproxen, tolmetin, sulindac, piroxicam, diflunisal, fenoprofen, or glucocorticoids; patients that have ingested corrosive chemicals; patients suffering from or at risk of aspiration pneumonia; or patients with a history of chronic or excessive alcohol consumption. BPI protein product treatment according to the invention is also contemplated for patients in intensive care situations, or pre-and/or post-operatively to prevent aspiration of gastric acid.

The invention further contemplates co-administration of BPI protein products with other therapeutic agents, such as antacids (e. g., magnesium carbonate or magnesium hydroxide or aluminum hydroxide), protective agents such as sucralfate or colloidal bismuth, nitrite scavengers (e. g., ascorbic acid or aminosulphonic acid), muscarinic cholinergic antagonists (e. g., prenzepine or telenzepine), prostaglandin agonists (e. g., misoprostol, 16,16-dimethyl prostaglandin E,, or other prostaglandin E, or E derivatives), carbenoxolone, H, histamine receptor antagonists (e. g., cimetidine, ranitidine, famotidine, nizatidine), or other H+/K} ATPase inhibitors (e. g., omeprazole, lansoprazole, pantoprazole, pariprazole or leminoprazole, or SCH 28080 or SK&F96067).

As used herein,"BPI protein product"includes naturally or recombinantly produced BPI protein; natural, synthetic, or recombinant biologically active polypeptide fragments of BPI protein; biologically active polypeptide variants of BPI protein or fragments thereof, including hybrid fusion proteins or dimers; biologically active polypeptide analogs of BPI protein or fragments or variants thereof, including cysteine-substituted analogs; or BPI- derived peptides. The BPI protein products administered according to this invention may be generated and/or isolated bv 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., 1 74: 649 (1991), or 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. Emmura. 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 (rBPI, 3) 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., sitpra, 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 I of Gray et au., supra, with the exceptions noted for rBPI,,, 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-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 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 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, or 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 cystine 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 cystine at residue number 132 is substituted with alanine and is designated rBPI,, Acys or rBPI,,. Production of this N- terminal analog of BPI, rBPI, In has been described in Horwitz et al.. Pnoteifr F. yression Puricarion. g : 2-40 (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) CI32A" or"rBPI (10-193) alfa''-'") 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 15, 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 BPl protein products include recombinantly- produced N-terminal analogs or fragments of BPI, especially those having a molecular weight of approximately between 20 to 25 kD such as rBPI,,, rBPI,,, rBPI (10-193) C132A, (rBPI (10-193) ala'3'), dimeric forms of these N-terminal polypeptides (e. g., rBPI42 dimer), or BPI-derived peptides. Particularly preferred BPI-derived peptides include XMP. 391 (SEQ ID NO: 4) or XMP. 416 (SEQ ID NO: 5).

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 (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 NaCI, pH 5.0) comprising 0.1 % by weight of poloxamer 188 (Pluronic F-68, BASF Wyandotte, Parsippany, NJ) and 0.002% by weight of polysorbate 80 (Tween 80, ICI Americas Inc., Wilmington. DE). Another stable pharmaceutical composition containing BPI protein products (e. g., rBPI,,) comprises the BPI protein product at a concentration of 2 mg ml in 5 mM citrate, 150 mM NaCI, 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. 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.

BPI protein product may also be administered in association (including covalent or non-covalent association) with targeting agents for delivery to specific cell types or tissues.

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 or retrobulbar, intrathecal, intraperitoneal (e. g. by intraperitoneal lavage), intrapulmonary (using powdered drug, or an aerosolized or nebulized drug solution), or transdermal.

When given parenterally, BPI protein product compositions are generally injecte in doses ranging from 1 llg/kg to 100 mg/kg per day, preferably at doses ranging from 0.1 m-g/kg to 20 mg/kg per day, more preferably at doses ranging from 1 to 20 mg/kg/day or 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, 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, co- pending U. S. Application Serial No. 08/557,289 and 08/557,287, both 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 uL of a BPI protein product composition may be applied one or more times per day as determined by the treating physician.

Those skilled in the art can readily optimize 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 patient.

"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. Example 1 addresses the inhibition of plasma membrane Ht/K-ATPase enzymic activity, as measured by reduction in phosphatase activity. Examples 2 and 3 address the inhibition of H4/K'ATPase activity in whole cells, as measured by a reduction in acid secretion into the medium. Examples 4-9 address isl vivo models of gastrointestinal inflammatory conditions that are exacerbated by gastric acidity, generally according to International Publication No. WO 96/01624.

EXAMPLE 1 Inhibition of ATPase enzymic activity in plasma membrane preparations y The effect of BPI protein products on H'/K--ATPase enzymic activity in mouse liver plasma membrane preparations was evaluated. Male laboratory mice (Mus nnrsculus) were sacrifice by cervical dislocation and their livers were immediately excised. The gall bladder and connective tissues were removed and the livers were washed in 0.25 M sucrose. Wet liver weight was determined after blotting the washed liver on absorbent paper. All subsequent steps of the fractionation procedure were performed on ice. The weighted livers were then homogenized with a Potter Elvehjem tissue homogenizer and six passes of a teflon pestle in 9 volumes of 0.25 M sucrose with protease inhibitors (2 ug/mL pepstatin A, 2 ug/mL aprotinin and ImM phenylmethylsulfonyl fluoride (PMSF). Liver homogenates were then centrifuged in a Beckman (Fullerton, CA) J2-21 centrifuge for 10 minutes at 600 x g. The 600 x g pellet (nuclei, unbroken cells and connective tissue) was discarded and the supernatant was subjected to further centrifugation at 7000 x g for 10 minutes.

The 7000 x g supernatant was centrifuged at 100000 x g for 60 minutes in a Beckman L5-50 ultracentrifuge to form the crude microsomal pellet and cytosol. The microsomal pellet was resuspended in 0. 25 M sucrose with protease inhibitors at a volume equal to the original wet liver weight.

The effect of various test compounds on the enzymic activity of the ATPase in the suspension was measured by the colonmetric determination of phosphate release, as follows. Assays were conducted in a 96-well plate. Each well contained approximately 10-15 uL (per lOOpL total volume) microsome suspension in incubation buffer (10 mM MES-Tris, pH mM NH4CI). Five pL of one of the following test compounds were added to give the indicated final concentration: 1.35 HM rBPI,,, 0. 37, uM omeprazole acid, 0. 37 pM omeprazole, 0. ? 5 uM oligomycin, 10 mgiml efrapeptin F&G, 0.015 uM sodium azide, 0.076 pM XMP. 416 (SEQ ID NO: 5) 0.082 uM XMP. 391 (SEQ ID NO: 4), 0.553, uM

XMP. 416 and 0.482 uM XMP. 391. Each plate contained an Enzyme Blank [test compound and buffer alone, without the ATPase-containing suspension], a Positive Control [ATPase-containing suspension alone, without test compound added], and a Phosphate Standard [ATPase-containing suspension containing 50 nM phosphate (5 ul of 10 mM NaH, PO,)].

The 96-well plate was incubated 10 min at 21°C (room temperature). The reaction was initiated by adding 50 1ll of ATP Stock solution [1 OmM MES, 15mM ATP, 15mM MgSO,, 25mM NH, C1,0.05% (w/v) deoxycholate, adjusted to pH 6.5 with Tris base] to each well. The plate was incubated for a total of 15 minutes. Plates were centrifuged in a Beckman J-6M centrifuge for 5 minutes at 1200 rpm. One hundred uL of each supernatant was transferred to a new 96-well plate. One hundred fil of Color Developing Reagent, a combined stop solution and color development reagent, was added [prepared by adding 0.5g Ascorbic acid to 30 ml H, O, followed by adding 5 ml 12% Ammonium Molybdate in 12N H. SO4 and 5 ml of 10% sodium lauryl sulfate, followed by adjusting total volume to 50 ml with H, O]. All reagents were added to each row at 30 second intervals to ensure that all samples were incubated for the identical length of time. The OD6>0nm of each well was determined using a Molecular Devices Vmax Kinetic Microplate Reader (Sunnyvale, CA), and the OD value for the Enzyme Blank was subtracted from each value.

Figure 1 shows the phosphatase activity results for the microsomal preparations; BPI protein products inhibited the ATPase activity of plasma membrane ATPase.

EXAMPLE 2 Inhibition of H+/K+ ATPase acid secreting activity in cells These experiments were designed to determine the ability of RAW 264.7 cells (a murine monocytic cell line) to acidify culture medium as they proliferate and metabolize. In these experiments, the medium is acidified after 24 hours with no decrease in cell viabilitv. This acidification is the result

of metabolic activity within the cells and subsequent pumping of the metabolic byproduct protons from the cell via the plasma membrane proton pump (a P-type H-/K--ATPase). Compounds that inhibit the acid secretion from mammalian cell lines were evaluated by measuring this medium acidification as follows.

The cells were plated into 48-well tissue culture plates at a cell density of 2.5 x 106 cells per well and allowed to become confluent for 24 hours.

The cells were plated in RPMI 1640 medium with Pen/Strep/Glutamine, 10 mM HEPES, 0.15M 2-mercaptoethanol and 10% Fetal Bovine Serum. This medium is normally buffered to approximately pH 7.4. Fresh medium was applied to the confluent cells and after 24 hours incubation, 1 mL culture supernatant was removed from each well and analyzed for pH using a Orion Research Digital Ion analyzer/501 (Beverly, MA) pH meter and Orion semi-micro microelectrode.

Toxic effects of the tested compounds were dissociated from inhibition of acid secretion by measuring cell proliferation in parallel 96-well microtiter plates with identical medium and identical inhibitor concentrations. Proliferation was measured by'H-thymidine incorporation over 24 hours using 2.5 x 10 5RAW 264.7 cells/well. Cells were harvested onto glass microfibre filters using an Inotech Cell Harvester (Lansing, MI) andH-thymidine incorporation was quantified on the dried filters using an Inotech Trace 96 Automatic Filter Counting System radioactivity counter. Results of pH and 3H-thyrnidine measurements for XMP. 416 are shown in Figure 2. Toxic effects of the peptide at concentrations greater than 50 pg/ml XMP. 416 are clearly dissociated from the inhibition of medium acidification at 12.5 tlg/ml XMP. 416.

EXAMPLE 3 Inhibition of H+/K+ ATPase acid secreting activity The inhibition of H-/K ATPase activity in gastnc vesicles is measured as follows according to U. S. Patent No. 5,420,135. Lyophilized gastric vesicles are prepared from pig fundic mucosa according to Keeling et. al., Bio- chem. Plrcr-ntacol., 34 : 2967 (1985). Potassium-stimulated ATPase activity is

determined at 37°C in the presence of the following: 10 mM PIPES/Tns buffer, pH 7.0,2 mM MgSO4,1 mM KCI, 2 mM Na, ATP and 3-6 llg protein/ml lyophilized gastric vesicles. After incubation for 30 minutes, the amount of inorganic phosphate hydrolysed from ATP is determined by the method of Yoda and Hekin, Biochem. Biophvs. Res. Commun., 40: 880 (1970).

Alternatively, the inhibition of acid secretion is evaluated by measuring'''C-aminopyrine accumulation by parietal cells according to U. S.

Patent No. 5,523,303. Gastric mucosal cells are prepared from rat stomach as follows. Wistar rats (130-r60 g) are killed by decapitation, the stomachs are rapidly excised and their contents washed out with saline. The stomachs are then everted and filled with 2.5 mg/ml of pronase-containing buffer. These sacs are incubated for 60 minutes at 37°C, in carbogen-gassed medium, followed by gentle stirring at room temperature for 45 minutes by a magnetic stirrer in order to dispense the cells from the mucosa of the everted stomachs digested only from the serosal side. The viability of the cells is determined by trypan-blue exclusion test, and the percentage of parietal cells is determined by their morphology.

Acid production by these gastric parietal cells prepared in this manner is induced by cyclic AMP, histamine (in the presence of 3-isobutyl-I- methylxanthine) or carbachol. Acid production in the presence and absence of inhibitors is assessed by measuring the accumulation of'''-C-aminopyrine. The undissociated weak base can penetrate into the acid-containing compartments of the cells. In the acidic compartment, the aminopyrine dissociates, after which it can no longer penetrate the membrane because the membrane is impermeable to the dissociated form. Thus, the distribution of"-C-aminopyrine between the extracellular and intracellular spaces is an indirect quantitative index for cellular acid production [Schepp et al., Ana. J. Phvsiol. 259, Gccstr-ointest. Liven Plysiol., 22: G646-654 (1990)].

EXAMPLE 4 Inhibition of gastric acid secretion Inhibition of gastric secretion is measured according to the method of Shay ligation, Gastroenterologv, 26: 903 (1954). Male Sprague-Dawley rats weighing 180-200g are starved for 24 hours and their pylorus is ligated. A BPI protein product or omeprazole as a positive control is administered. Four hours later, the stomach is removed, and the acidity and amount of gastric juice is measured. The inhibition of gastric secretion is calculated by comparing the measured values with those of the control group to which no test compound was administered. The ED50 of the test compound is the dose that inhibits the gastric secretion by 50%.

EXAMPLE 5 Protection against ethanol-induced gastric ulcers The protective effect against the formation of ethanol-induced gastric ulcerative lesions is measured generally according to Robert, Gastroenterologv, 77: 761-767 (1979). Male Sprague-Dawley rats weighing 180- 200g are starved for 24 hours. A BPI protein product or omeprazole as a positive control are administered. Thirty minutes later, 5 ml/kg absolute ethanol is orally administered to produce an erosion of the stomach wall. Ninety minutes later, the stomach is removed, and the length, frequency and degree of the ulcerative lesions is measured. The measured values are compared with those of the control group to which ho test compound was administered, and the ED50 of the test compound which inhibits the lesion by 50% is calculated. Alternatively, the percentage inhibition of lesion formation may be calculated.

EXAMPLE 6 Protection against mepirozole-induced duodenal ulcers The protective effect against the formation of mepirozole-induced duodenal ulcerative lesions is measured as follows. Male sprague-Dawley rats

weighing 200-230g are not starved, and a BPI protein product or omeprazole as a positive control is administered. Thirty minutes later, 250mg/kg mepirizole suspended in 1% CMC is orally administered, and the rats are starved for 24 hours. The duodenum of each rat is removed and the degree of the ulceration is measured. The EDso of the test compound which inhibits the ulcer by 50% is calculated.

EXAMPLE 7 Protection against indomethacin-induced gastric lesions The protective effect against the formation of indomethacin- induced gastric lesions is measured as follows. Male Sprague-Dawley rats are starved for 48 hours and prohibited from access to water for 2 hours. A BPI protein product or omeprazole as a positive control is administered, and 35 mg/kg of indomethacin is subcutaneously administered to cause gastric lesions. The ED50 o of the test compound which inhibits the lesions by 50% is calculated.

EXAMPLE 8 Protection against stress-induced ulcers Stress is an important factor in causing gastric lesions. The protective effect against the formation of stress-induced ulcers is evaluated as follows. Male Sprague-Dawley rats are starved for 24 hours prior to carrying out the experiment, in which the rats are stressed by immersing them in water. Thirty minutes prior to immersing the rats in water, a BPI protein product or omeprazole as a positive control is administered. The EDso of the test compound which inhibits the lesions by 50% is calculated.

EXAMPLE 9 Healing of acetic acid-induced ulcers The effect on heating of acetic acid-induced ulcerative lesions is evaluated as follows. Male sprarue-Dawles rats are starved for 5 hours. 20 Lit

of 30% acetic acid is injected into the submucosal layer of the stomach using a microsyringe, to induce a circular ulcer on the stomach wall. Various doses of BPI protein product or omeprazole as a positive control are administered for 10 days, and the healing of the ulcer is monitored. The percentages of the healing of the ulcer are calculated and compare to the control group that received no test compound.

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 appende claims should be placed thereon.