WO2001003724A1 - Therapeutic uses of bpi protein products for inhibiting h+/k+ atpase activity - Google Patents

Therapeutic uses of bpi protein products for inhibiting h+/k+ atpase activity Download PDF

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WO2001003724A1
WO2001003724A1 PCT/US2000/009125 US0009125W WO0103724A1 WO 2001003724 A1 WO2001003724 A1 WO 2001003724A1 US 0009125 W US0009125 W US 0009125W WO 0103724 A1 WO0103724 A1 WO 0103724A1
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bpi
bpi protein
mammal
gastπc
protein product
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PCT/US2000/009125
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French (fr)
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Roger G. Little
Susan Abrahamson
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Xoma Technology Ltd.
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Priority to AU42042/00A priority Critical patent/AU4204200A/en
Publication of WO2001003724A1 publication Critical patent/WO2001003724A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4742—Bactericidal/Permeability-increasing protein [BPI]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1751—Bactericidal/permeability-increasing protein [BPI]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants

Definitions

  • the present invention relates generally to novel therapeutic uses of BPI protein products that involve inhibition of adenosine triphosphatase (ATPase) activity in mammals.
  • ATPase adenosine triphosphatase
  • H7K7-ATPases are present in cytoplasmic membranes of eukaryotic cells and act as proton pumps.
  • an H7K + -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 H7K ' -ATPase is also expressed in kidneys. In the parietal cell, its function is to secrete acid by forcing an exchange of H 3 O * for K " . In the kidneys it is probably responsible for a small part of the elimination of an acid load, whereas the homologous colonic H7KT-ATPase is likely involved in potassium homeostasis.
  • Gastric H7K - 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.
  • P-type ATPases include the yeast H7K -ATPase (which regulates intracellular pH, ion balance and nut ⁇ ent uptake by the cell), the mammalian Na ' K " ATPases ⁇ e.g., in kidney) and the mammalian Ca 2* -ATPase (e g , muscle)
  • yeast H7K -ATPase which regulates intracellular pH, ion balance and nut ⁇ ent uptake by the cell
  • mammalian Na ' K " ATPases ⁇ e.g., in kidney
  • Ca 2* -ATPase e g , muscle
  • the family of P-type ATPases shares little homology with the distinct family of F 0 -F, ATPases, which includes the H7K ⁇ - ATPases of bacte ⁇ a, mitochond ⁇ a and chloroplasts and the ATP synthases from mitochond ⁇ a. [Serrano et al, Nature, 319.689-693 (1986) ]
  • omeprazole a py ⁇ d ⁇ nyl-2- methylenesulf ⁇ nyl-2 benzimidazole de ⁇ vative
  • aceprazole acts by inhibiting the gast ⁇ c pa ⁇ etal cell H7K " ATPase
  • This enzyme plays an important role in the actual secretion of HCl Blockade of the pump inhibits acid secretion regardless of the pathway of stimulation that is being used by the cell.
  • peptic ulcer disease gast ⁇ c and duodenal ulcer disease
  • drugs such as aspirin, non- steroidal anti-inflammatory drugs (NSAIDs), e g , lndomethacin, lbuprofen, naproxen, tolmetin, suhndac, piroxicam, diflunisal, fenoprofen.
  • NSAIDs non- steroidal anti-inflammatory drugs
  • Peptic ulcers are a part of Zollinger-EIhson syndrome, which is characterized by gast ⁇ c acid j - hypersecretion caused by a gast ⁇ n-secreting tumor of the pancreatic islet cells
  • Helicobacter pvlon infection is frequently present in patients with peptic ulcer disease and has been proposed as a cont ⁇ butory or modifying factor therefor
  • eosinophilic gast ⁇ tis, granulomatous gast ⁇ tis and p ⁇ or gast ⁇ c surgery may produce gast ⁇ tis or gast ⁇ c ulceration [Har ⁇ son's P ⁇ nciples of Internal Medicine, 13th ed , Isselbacher et al , eds , McGraw-Hill, NY (1994), pages 1363-1382 ]
  • H 2 histamine receptor antagonists e g , cimetidine, ramtidme, famotidme, nizatidme
  • covalent inhibitors of the H7K -ATPase of the pa ⁇ etal cell e g , omeprazole, lansoprazole
  • Protective agents such as sucralfate, colloidal bismuth, prostaglandm agonists (such as misoprostol or other prostaglandm E, and E 2 de ⁇ vatives) and antacids are also effective Outside of the United States, musca ⁇ mc chohnergic antagonists (e g , prenzepine and telenzepine) and carbenoxolone have been used to treat peptic ulcer disease [Goodman & Gilman, The Pharmacological Basis of Therapeutics, 9th
  • 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
  • PMNs or neutrophils mammalian polymorphonuclear leukocytes
  • BPI protein products are bacte ⁇ cidal for gram-negative bacte ⁇ a, as desc ⁇ bed 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 suffe ⁇ ng 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 bacte ⁇ al infections, as described m
  • BPI protein products are also bacte ⁇ cidal for gram-positive bactena and mycoplasma, and enhance the effectiveness of antibiotics in
  • BPI protein products exhibit anti-protozoan activity, as desc ⁇ bed in U S Patent Nos. 5,646,1 14 and 6,013,629 and International Pub cationNo WO 96/01647 (PCT/US95/08624).
  • BPI protein products exhibit anti-chlamydial activity, as desc ⁇ bed in co-owned U.S Patent No 5,888,973 and WO 98/06415 (PCT/US97/13810).
  • BPI protein products exhibit anti-mycobacte ⁇ al activity, as described in co-owned, co-pending U.S. Application Se ⁇ al No. 08/626,646, which is in turn a continuation of U.S. Application Se ⁇ al No 08/285,803, which is in turn a continuation-m-part ofU.S. Application Se ⁇ al No. 08/031,145 and corresponding International Publication No. WO 94/20129 (PCT/US94/02463)
  • BPI protein products are also useful for treatment of specific disease conditions, such as memngococcemia in humans (as described in U.S Patent Nos 5,888,977 and 5,990,086 and International Publication No WO97/42966 (PCT/US97/08016), hemorrhage due to trauma in humans, (as desc ⁇ bed in U S Patent Nos.
  • BPI protein products are also useful in antithrombotic methods, as desc ⁇ bed m U S Patent Nos 5,741,779 and 5,935,930 and corresponding International Publication No WO 97/42967 (PCT/US7/08017)
  • the present invention provides novel therapeutic uses for BPI protein products, including BPI-de ⁇ ved peptides, that involve inhibition of H7K ATPase activity, including methods for inhibiting gast ⁇ c acid secretion
  • 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).
  • GSD gastroesophageal reflux disease
  • One aspect of the invention provides a method of inhibiting H /K ATPase activity in a mammal in need thereof comp ⁇ sing administe ⁇ ng to said mammal a therapeutically effective amount of a BPI protein product
  • Another aspect of the invention provides a method of inhibiting gast ⁇ c acid secretion in a mammal in need thereof comp ⁇ sing administe ⁇ ng 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 2] , rBPI 2 resort rBPI(10- 193)C132A, (rBPI(10-193)ala 132 ), dime ⁇ c forms of these N-terminal polypeptides (e.g., rBPI 42 dimer), or BPI-de ⁇ ved peptides.
  • Exemplary BPI-de ⁇ ved peptides include XMP.391 (SEQ ID NO" 4), XMP.416 (SEQ LD NO: 5) or XMP 445 (SEQ ID NO: 6) [the structure and activity of which are desc ⁇ bed m co-owned, co-pending U.S. Se ⁇ al No. U S. Se ⁇ al No. 09/406,243 filed September 24, 1999, incorporated herein by reference]
  • a BPI protein product may be accompanied by the concurrent administration of other therapeutic agents, such as agents that also inhibit gast ⁇ c acid secretion, protect the gast ⁇ c mucosa, or neutralize gast ⁇ c acids It is also contemplated that a BPI protein product may be concurrently administered with agents that tend to induce gast ⁇ c injury, such as aspi ⁇ n, NSAIDs, glucocorticoids, or alcohol.
  • agents that tend to induce gast ⁇ c injury such as aspi ⁇ n, NSAIDs, glucocorticoids, or alcohol.
  • the invention also provides methods of screening BPI protein products, including BPI-de ⁇ ved peptides. for inhibition of H /K ATPase activity
  • Such methods would comp ⁇ se steps of, e g , contacting an H /K ATPase with a BPI protein product and measu ⁇ ng 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 a .
  • throu g h detecting acidification of medium or anv one of the other assays desc ⁇ bed herein
  • the screening methods involve a further step of testing candidates in animal models of gastrointestinal inflammatory conditions that are exacerbated by gast ⁇ c acidity
  • 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 3 H-thym ⁇ dme measurements in a culture of whole cells.
  • the present invention provides novel therapeutic uses for BPI protein products, particularly BPI-de ⁇ ved peptides, that involve inhibition of H7K ATPase activity, or methods for inhibiting acid secretion, including acid secretion by gast ⁇ c pa ⁇ etal cells, or methods for increasing potassium excretion
  • 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
  • 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 gast ⁇ c acidity, including, for example, gastroesophageal reflux disease (GERD), esophagitis, gastritis, duodenitis.
  • GFD gastroesophageal reflux disease
  • esophagitis esophagitis
  • gastritis e.g., duodenitis.
  • BPI protein product treatment is contemplated for patients being treated with drugs that induce gast ⁇ c injury, such as aspi ⁇ n, non-steroidal anti-inflammatory drugs (NSAIDs), e g , mdomethacin, lbuprofen, naproxen, tolmetin, sulmdac, piroxicam, diflunisal, fenoprofen, or glucocorticoids, patients that have ingested corrosive chemicals, patients suffe ⁇ ng from or at ⁇ sk of aspiration pneumonia, or patients with a history oT 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 gast
  • drugs that induce gast ⁇ c injury such as aspi ⁇ n, non-steroidal anti-inflammatory drugs (NSAIDs), e g , mdomethacin, lbuprofen, naproxen, tolmet
  • the invention further contemplates co-admmistration 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 aminosulphomc acid), musca ⁇ mc chohnergic antagonists (e g , prenzepine or telenzepine), prostaglandm agonists (e g , misoprostol, 16,16-d ⁇ methyl prostaglandm E 2 , or other prostaglandm E, or ⁇ de ⁇ vatives), carbenoxolone, H 2 histamine receptor antagonists (e g , cimetidine, ranitidine, famotidine, mzatidme), or other H7K ⁇ ATPase inhibitors (e g , omeprazole, lanso
  • BPI protein product includes naturally or recombinantly produced BPI protein, natural, synthetic, or recombinant biologically active polypeptide fragments of BPI protein, biologically active polypeptide va ⁇ ants 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- de ⁇ v ed 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 (PC
  • BPI fragments include biologically active molecules that have the same or similar ammo acid sequence as a natural human BPI holoprotein, except that the fragment molecule lacks amino-terminal ammo acids, internal ammo acids, and/or carboxy-termmal ammo acids of the holoprotein, including those desc ⁇ bed in U S Patent Nos 5,198,541 and 5,641,874
  • Nonhmiting examples of such fragments include an N-terminal fragment of natural human BPI of approximately 25 kD, desc ⁇ bed in Ooi et al , J Exp Med 174 649 (1991), or the recombinant expression product of DNA encoding N-terminal amino acids from 1 to about 193 to 199 of natural human BPI, desc ⁇ bed in Gazzano-Santoro et al , Infect Immun 60 4754-4761 (1992).
  • rBPI 23 an expression vector was used as a source of DNA encoding a recombinant expression product (rBPI 23 ) having the 31 -residue signal sequence and the first 199 amino acids of the N-terminus of the mature human BPI, as set out in Figure 1 of Gray et al , supra, except that vahne 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 rBPL, and with the exception that residue 417 is alanine (specified by GCT) rather than vahne (specified by GTT)
  • Another fragment consisting of residues 10-193 of BPI has been desc ⁇ bed in U S Patent No 6,013,631 ,
  • 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 hyb ⁇ d fusion proteins comp ⁇ sing, at the amino-term al 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 allehc va ⁇ ant thereof
  • B ⁇ olog ⁇ call> active analogs of BPI include but are not limited to BPI protein products wherein one or more ammo acid residues have been replaced by a different amino acid
  • BPI analogs include but are not limited to BPI protein products wherein one or more ammo acid residues have been replaced by a different amino acid
  • 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 ammo acid
  • a stable BPI protein product desc ⁇ bed by this application is the expression product of DNA encoding from ammo 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 rBPK, ⁇
  • BPI protein products are preferably accomplished with a pharmaceutical composition comp ⁇ sing a BPI protein product and a pharmaceutically acceptable diluent, adjuvant, or earner
  • 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 , rBPI 23 ) comp ⁇ ses the BPI protein product at a concentration of 1 mg/ml in citrate buffered saline (5 or 20 mM citrate, 150 mM NaCl, pH 5 0) comp ⁇ smg 0 1% by weight of poloxamer 188 (Pluromc F-68, BASF Wyandotte, Parsippany, NJ) and 0 002%.
  • 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 comp ⁇ sing 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, intrape ⁇ toneal (e g by intrapentoneal lavage), mtrapulmonary (using powdered drug, or an aerosolized or nebulized drug solution), or transdermal
  • BPI protein product compositions are generally injected in doses ranging from 1 ⁇ g/kg to 100 mg/kg per day, preferably at doses ranging from 0 1 mg/kg to 20 mg kg per day, more preferably at doses ranging from 1 to 20 mg/kg/day 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
  • BPI protein products are preferably administered by an initial b ⁇ ef infusion followed by a continuous infusion
  • the preferred intravenous regimen is a 1 to 20 mg/kg b ⁇ ef 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 dosmg regimen is a 1 to 4 mg/kg initial b ⁇ ef
  • Topical routes include administration in the form of salves, creams, jellies, ophthalmic drops or ointments (as desc ⁇ bed 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
  • topical administration in drop form about 10 to 200 ⁇ L of a BPI protein product composition may be applied one or more times per day as determined by the treating physician
  • 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
  • the BPI protein product may be administered intravenously
  • Example 1 addresses the inhibition of plasma membrane H7K ATPase enzymic activity, as measured by reduction in phosphatase activity
  • Examples 2 and 3 address the inhibition of H /K ATPase activity m whole cells, as measured by a reduction in acid secretion into the medium
  • Examples 4-9 address in ⁇ vo models of gastrointestinal inflammatory conditions that are exacerbated by gast ⁇ c acidity, generally according to International Publication No WO 96/01624 EXAMPLE 1
  • mice Male laboratory mice (Mas musculus) were sac ⁇ ficed 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 homogemzer and six passes of a teflon pestle in 9 volumes of 0 25 M sucrose with protease inhibitors (2 ⁇ g/mL pepstatin A, 2 ⁇ g/mL aprotimn and lmM phenylmethylsulfonyl fluoride (PMSF) Liver homogenates were then centrifuged in a Beckman (Fullerton, CA) J2-21 centrifuge for 10 minutes at
  • the 7000 x g supernatant was centrifuged at 100000 x g for 60 minutes in a Beckman L5-50 ultracent ⁇ fuge 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 96-well plate was incubated 10 min at 21°C (room temperature) The reaction was initiated by adding 50 ⁇ l of ATP Stock solution [lOmM MES, 15mM ATP, 15mM MgSO 4 , 25mM NH 4 C1, 0 05% (w/v) deoxycholate, adjusted to pH 6 5 with Tns base] to each well The plate was incubated for a total of 15 minutes Plates were cent ⁇ fuged in a Beckman J-6M cent ⁇ fuge for 5 minutes at 1200 rpm One hundred ⁇ L of each supernatant was transferred to a new 96-well plate One hundred ⁇ l 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 2 O, followed by adding 5 ml 12% Ammonium Molybdate in 12N H 2 SO 4 and 5 ml of 10% sodium lauryl sulfate, followed by adjusting total volume to 50
  • Figure 1 shows the phosphatase activity results for the microsomal preparations, BPI protein products inhibited the ATPase activity of plasma membrane ATPase
  • the inhibition of acid secretion is evaluated by measu ⁇ ng l4 C-am ⁇ nopy ⁇ ne accumulation by parietal cells according to U S Patent No 5,523,303 Gast ⁇ c mucosal cells are prepared from rat stomach as follows Wistar rats (130-T60 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 stir ⁇ ng 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 pa ⁇ etal cells is determined by their morphology
  • Inhibition of gast ⁇ c secretion is measured according to the method of Shay ligation, Gastroenterologx , 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 gast ⁇ c juice is measured The inhibition of gast ⁇ c secretion is calculated by compa ⁇ ng the measured values with those of the control group to which no test compound was administered The ED S0 of the test compound is the dose that inhibits the gast ⁇ c secretion by 50%>
  • the protective effect against the formation of ethanol-induced gastric ulcerative lesions is measured generally according to Robert, Gastroenterologv, 11 761 -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
  • 5 ml/kg absolute ethanol is orally administered to produce an erosion of the stomach wall
  • 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 fio test compound was administered, and the ED ⁇ 0 of the test compound which inhibits the lesion by 50% is calculated Alternatively, the percentage inhibition of lesion formation may be calculated
  • the protectiv e effect against the formation of mepirozole-induced duodenal ulcerative lesions is measured as follows Male sprague-Daw ley 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 mepi ⁇ zole 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 ED, 0 of the test compound which inhibits the ulcer by 50% is calculated
  • the protective effect against the formation of indomethac - induced gast ⁇ c 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 indomefhacm is subcutaneously administered to cause gast ⁇ c lesions The ED S0 of the test compound which inhibits the lesions by 50% is calculated
  • acetic acid-induced ulcerative lesions The effect on healing of acetic acid-induced ulcerative lesions is evaluated as follows Male sprague-Dawlev rats are starved for 5 hours 20 ul of 30% acetic acid is injected into the submucosal layer of the stomach using a microsy ⁇ nge, 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 compared to the control group that received no test compound

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Abstract

New therapeutic uses for BPI protein products that involve inhibition of H+/K+ ATPase activity, including inhibiting gastric acid secretion, are described.

Description

THERAPEUTIC USES OF BPI PROTEIN PRODUCTS FOR INHIBITING H7K+ ATPASE ACTIVITY
This application claims priority of U.S. Provisional Application No. 60/143,374 filed July 12, 1999 [Docket No. 27129/35826], the disclosure of which is incorporated herein by reference., the disclosure of which is incorporated herein by reference.
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
H7K7-ATPases are present in cytoplasmic membranes of eukaryotic cells and act as proton pumps. In particular, an H7K+-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 H7K'-ATPase is also expressed in kidneys. In the parietal cell, its function is to secrete acid by forcing an exchange of H3O* for K". In the kidneys it is probably responsible for a small part of the elimination of an acid load, whereas the homologous colonic H7KT-ATPase is likely involved in potassium homeostasis. Gastric H7K - 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, Pharmacotherapy, 17:22-27 ( 1997).] Other members of the extended family of P-type ATPases include the yeast H7K -ATPase (which regulates intracellular pH, ion balance and nutπent uptake by the cell), the mammalian Na' K" ATPases {e.g., in kidney) and the mammalian Ca2*-ATPase (e g ,
Figure imgf000003_0001
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 pπmary 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 Perhn, Crit Re\ Microbwl, 20.209-223-(1994) ] The family of P-type ATPases shares little homology with the distinct family of F0-F, ATPases, which includes the H7K~- ATPases of bacteπa, mitochondπa and chloroplasts and the ATP synthases from mitochondπa. [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 pyπdιnyl-2- methylenesulfιnyl-2 benzimidazole deπvative) acts by inhibiting the gastπc paπetal cell H7K" ATPase This enzyme plays an important role in the actual secretion of HCl 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 (gastπc and duodenal) ulcer disease is approximately 10%, and some physicians estimate that 50%o 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 gastπc cancers, by drugs such as aspirin, non- steroidal anti-inflammatory drugs (NSAIDs), e g , lndomethacin, lbuprofen, naproxen, tolmetin, suhndac, 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-EIhson syndrome, which is characterized by gastπc acid j - hypersecretion caused by a gastπn-secreting tumor of the pancreatic islet cells Helicobacter pvlon infection is frequently present in patients with peptic ulcer disease and has been proposed as a contπbutory or modifying factor therefor In addition, eosinophilic gastπtis, granulomatous gastπtis and pπor gastπc surgery may produce gastπtis or gastπc ulceration [Harπson's Pπnciples 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 gastπc acidity effectively promote healing Such drugs include H2 histamine receptor antagonists (e g , cimetidine, ramtidme, famotidme, nizatidme) and covalent inhibitors of the H7K -ATPase of the paπetal cell (e g , omeprazole, lansoprazole) Protective agents such as sucralfate, colloidal bismuth, prostaglandm agonists (such as misoprostol or other prostaglandm E, and E2 deπvatives) and antacids are also effective Outside of the United States, muscaπmc chohnergic 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 gastπc H7K -ATPase antagonists include the K -competitive drugs SCH 28080 and SK&F96067 [Monk and Perhn, Crit Rev Microbwl , 20 209-223 (1994) ]
However, there remains a need for agents which are H7K - 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 Chem 254 1 1000 (1979)] or £ coli affimtv 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 bacteπcidal activity against a broad spectrum of gram-negative bacteria The molecular weight of human BPI is approximately 55,000 daltons (55 kD) The ammo 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 Chem 264 9505 ( 1989), incorporated herein by reference The Gray et al nucleic acid and amino acid sequence are set out m 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 lysme (specified by AAG) and residue 417 is alanine (specified by GCT) rather than valme (specified by GTT) BPI is a strongly cationic protein The N-terminal half of BPI accounts for the high net positive charge, the C-termmal 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-bacteπal efficacy of the naturally-deπved 55 kD human BPI holoprotein [Ooi et al , J Bio Chem 262 14891 - 14894 (1987)] In contrast to the N-terminal portion, the C-terminal region of the isolated human BPI protein displays only slightly detectable 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 "rBPI2„" has been produced by recombinant means and also retains anti- bacterial activity against gram-negativ e organisms [Gazzano-Santoro et al , Infect Immim 60 4754-4761 ( 1992) ] An N-terminal analog designated rBPlΗ (also referred to as rBPI( l -193)ala' ^) has been descπbed in U S Patent No 5,420,019
The bacteπcidal effect of BPI was oπginally reported to be highly specific to gram-negative species, e g , in Elsbach and W eiss. Inflammation - 3 -
Basic Principles and Clinical Con elates, eds Galhn et al , Chapter 30. Raven Press, Ltd (1992) The precise mechanism by which BPI kills gram-negative bacteπa 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 phosphohpids and peptidoglycans, alteπng 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, l 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 vaπety of beneficial activities
BPI protein products are bacteπcidal for gram-negative bacteπa, as descπbed 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 suffeπng 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 bacteπal infections, as described m 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/US94/1 1255) BPI protein products are also bacteπcidal for gram-positive bactena and mycoplasma, and enhance the effectiveness of antibiotics in gram-positive bacterial infections, as descπbed in U S Patent Nos 5,578,572 and 5,783,561 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 5,627,153 and International Publication No WO 95/19179 (PCT/US95/00498), and further as descπbed for BPI-denved peptides in U.S Patent No 5,858,974, which is in tum a continuation-m-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 descπbed in U S Patent Nos. 5,646,1 14 and 6,013,629 and International Pub cationNo WO 96/01647 (PCT/US95/08624). BPI protein products exhibit anti-chlamydial activity, as descπbed in co-owned U.S Patent No 5,888,973 and WO 98/06415 (PCT/US97/13810). Finally, BPI protein products exhibit anti-mycobacteπal activity, as described in co-owned, co-pending U.S. Application Seπal No. 08/626,646, which is in turn a continuation of U.S. Application Seπal No 08/285,803, which is in turn a continuation-m-part ofU.S. Application Seπal 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 m circulation, including effects on TNF, IL-6 and endotoxin are descπbed in U.S Patent Nos 5,643,875, 5,753,620 and 5,952,302 and corresponding International Publication No WO 95/19784 (PCT/US95/01 151 )
BPI protein products are also useful for treatment of specific disease conditions, such as memngococcemia in humans (as described in U.S Patent Nos 5,888,977 and 5,990,086 and International Publication No WO97/42966 (PCT/US97/08016), hemorrhage due to trauma in humans, (as descπbed 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 descπbed in U S Patent No 5,494,896 and corresponding International Publication No WO 96/30037 (PCT/US96/02349)) ischemia'reperfusion injury (as descπbed m U S Patent No 5,578,568), and depressed RES/hver resection (as descπbed in co-owned, co-pending U S Apphcation Seπal No 08/582,230 which is in turn a continuation of U S Application Seπal No 08/318,357, which is in turn a continuation-in-part of U S Application Seπal 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 hepaπn, as descπbed in U S Patent No 5,348,942, neutralize hepaπn in vitro as descπbed lπ U S Patent No 5,854,214, and are useful for treating chronic inflammatory diseases such as rheumatoid and reactive arthritis, for inhibiting endothehal cell proliferation, and for inhibiting angiogenesis and for treating angiogenesis-associated disorders including malignant tumors, ocular retinopathy and endometπosis, as descπbed 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 descπbed m 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-deπved peptides, that involve inhibition of H7K ATPase activity, including methods for inhibiting gastπc 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, gastπtis, duodenitis, gastric cancers, gastπnomas, Zollinger-El son syndrome, acute upper gastrointestinal bleeding, gastπc ulcers, duodenal ulcers, ingestion of corrosive chemicals stress ulcers, aspiration pneumonia, chronic or excessive alcohol consumption, patients in intensive care situations, or pre- and/or post- operatively to prevent aspiration of gastπc acid
One aspect of the invention provides a method of inhibiting H /K ATPase activity in a mammal in need thereof compπsing administeπng to said mammal a therapeutically effective amount of a BPI protein product
Another aspect of the invention provides a method of inhibiting gastπc acid secretion in a mammal in need thereof compπsing administeπng 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 rBPI2], rBPI2„ rBPI(10- 193)C132A, (rBPI(10-193)ala132), dimeπc forms of these N-terminal polypeptides (e.g., rBPI42 dimer), or BPI-deπved peptides. Exemplary BPI-deπved peptides include XMP.391 (SEQ ID NO" 4), XMP.416 (SEQ LD NO: 5) or XMP 445 (SEQ ID NO: 6) [the structure and activity of which are descπbed m co-owned, co-pending U.S. Seπal No. U S. Seπal 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 gastπc acid secretion, protect the gastπc mucosa, or neutralize gastπc acids It is also contemplated that a BPI protein product may be concurrently administered with agents that tend to induce gastπc injury, such as aspiπn, NSAIDs, glucocorticoids, or alcohol.
The invention also provides methods of screening BPI protein products, including BPI-deπved peptides. for inhibition of H /K ATPase activity Such methods would compπse steps of, e g , contacting an H /K ATPase with a BPI protein product and measuπng 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 a . through detecting acidification of medium or anv one of the other assays descπbed herein Optionally the screening methods involve a further step of testing candidates in animal models of gastrointestinal inflammatory conditions that are exacerbated by gastπc acidity
Numerous additional aspects and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed descπption 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-thymιdme 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-deπved peptides, that involve inhibition of H7K ATPase activity, or methods for inhibiting acid secretion, including acid secretion by gastπc paπetal cells, or methods for increasing potassium excretion The invention is based on the novel discovery that BPI protein products inhibit the H /Kτ 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 gastπc acidity, including, for example, gastroesophageal reflux disease (GERD), esophagitis, gastritis, duodenitis. gastπnomas, Zolhnger-Elhson syndrome, acute upper gastrointestinal bleeding, gastπc ulcers, duodenal ulcers or stress ulcers, including gastπtis or ulcer conditions not involving persistent Helicobacter infection Furthermore, BPI protein product treatment is contemplated for patients being treated with drugs that induce gastπc injury, such as aspiπn, non-steroidal anti-inflammatory drugs (NSAIDs), e g , mdomethacin, lbuprofen, naproxen, tolmetin, sulmdac, piroxicam, diflunisal, fenoprofen, or glucocorticoids, patients that have ingested corrosive chemicals, patients suffeπng from or at πsk of aspiration pneumonia, or patients with a history oT 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 gastπc acid
The invention further contemplates co-admmistration 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 aminosulphomc acid), muscaπmc chohnergic antagonists (e g , prenzepine or telenzepine), prostaglandm agonists (e g , misoprostol, 16,16-dιmethyl prostaglandm E2, or other prostaglandm E, or ϋ deπvatives), carbenoxolone, H2 histamine receptor antagonists (e g , cimetidine, ranitidine, famotidine, mzatidme), or other H7K~ ATPase inhibitors (e g , omeprazole, lansoprazole, pantoprazole, paπprazole or lem oprazole, 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 vaπants 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- deπv ed 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 puπfication 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 ammo acid sequence as a natural human BPI holoprotein, except that the fragment molecule lacks amino-terminal ammo acids, internal ammo acids, and/or carboxy-termmal ammo acids of the holoprotein, including those descπbed in U S Patent Nos 5,198,541 and 5,641,874 Nonhmiting examples of such fragments include an N-terminal fragment of natural human BPI of approximately 25 kD, descπbed in Ooi et al , J Exp Med 174 649 (1991), or the recombinant expression product of DNA encoding N-terminal amino acids from 1 to about 193 to 199 of natural human BPI, descπbed in Gazzano-Santoro et al , Infect Immun 60 4754-4761 (1992). and referred to as rBPI23 In that publication, an expression vector was used as a source of DNA encoding a recombinant expression product (rBPI23) having the 31 -residue signal sequence and the first 199 amino acids of the N-terminus of the mature human BPI, as set out in Figure 1 of Gray et al , supra, except that vahne 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 rBPL, and with the exception that residue 417 is alanine (specified by GCT) rather than vahne (specified by GTT) Another fragment consisting of residues 10-193 of BPI has been descπbed in U S Patent No 6,013,631 , continuation-in- part U S Application Seπal 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 dimeπc forms of BPI fragments, as descπbed 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 vaπants of BPI (BPI vaπants) include but are not limited to recombinant hybπd fusion proteins, compπsing BPI holoprotein or biologically active fragment thereof and at least a portion of at least one other polypeptide, or dimenc forms of BPI vaπants Examples of such hybπd fusion proteins and dimeπc forms are descπbed 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 hybπd fusion proteins compπsing, at the amino-term al 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 allehc vaπant thereof
Bιologιcall> active analogs of BPI (BPI analogs) include but are not limited to BPI protein products wherein one or more ammo 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 ammo acid A stable BPI protein product descπbed by this application is the expression product of DNA encoding from ammo 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 rBPK,Δcys or rBP , Production of this N- ter inal analog of BPI, rBPI: ! , has been described in Horwitz et al . Protein Expi ession Purification 5 28-40 ( 1996) Similarly, an analog consisting of residues 10-193 of BPI in which the cysteine at position 132 is replaced with an alamne (designated "rBPI(10-193)C132A" or "rBPI(10-193)alalj2") has been described in U S Patent No 6,013,631, continuation-in-part U S Application Seπal 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 dimeπc 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 deπved from or based on BPI produced by synthetic or recombinant means (BPI-deπved peptides), such as those descπbed in International Publication No WO 97/04008 (PCT/US96/03845), which corresponds to U S Application Seπal 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 WO94/20532 (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 Seπal No 08/183,222, filed January 14, 1994, which is a contmuation-in-part of U S Application Seπal 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 bv reference Presently preferred BPI protein products include recombinantly- produced N-termmal analogs or fragments of BPI, especially those having a molecular weight of approximately between 20 to 25 kD such as rBPI21, rBPI21, rBPI(10-193)C132A, (rBPI(10-193)ala132), dimeπc forms of these N-terminal polypeptides (e g , rBPI42 dimer), or BPI-deπved peptides Particularly preferred BPI-deπved 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 compπsing a BPI protein product and a pharmaceutically acceptable diluent, adjuvant, or earner 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) compπses the BPI protein product at a concentration of 1 mg/ml in citrate buffered saline (5 or 20 mM citrate, 150 mM NaCl, pH 5 0) compπsmg 0 1% by weight of poloxamer 188 (Pluromc F-68, BASF Wyandotte, Parsippany, NJ) and 0 002%. by weight of polysorbate 80 (Tween 80, ICI Ameπcas Inc , Wilmington. DE) Another stable pharmaceutical composition containing BPI protein products (e g , rBPI2l) compπses the BPI protein product at a concentration of 2 mg/ml in 5 mM citrate, 150 mM NaCl, 0 2% poloxamer 188 and 0 002%> polysorbate 80 Such preferred combinations are descπbed 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 descπbed in U S Patent No 5,912,228 and corresponding International Publication No WO96/21436 (PCT7US96/01095), all of which are incorporated herein by reference, other poloxamer formulations of BPI protein products with enhanced acti ity 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 compπsing 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, intrapeπtoneal (e g by intrapentoneal lavage), mtrapulmonary (using powdered drug, or an aerosolized or nebulized drug solution), or transdermal
When given parenterally, BPI protein product compositions are generally injected in doses ranging from 1 μg/kg to 100 mg/kg per day, preferably at doses ranging from 0 1 mg/kg to 20 mg kg per day, more preferably at doses ranging from 1 to 20 mg/kg/day 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 bπef infusion followed by a continuous infusion The preferred intravenous regimen is a 1 to 20 mg/kg bπef 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 dosmg regimen is a 1 to 4 mg/kg initial bπef 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 descπbed 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 μL 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 compπsing 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) ιs(are) administered intravenously, intramuscularly, subcutaneously, orally or intrapeπtoneally 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 m a special form for gastπc delivery, while the second agent(s) ιs(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 H7K ATPase enzymic activity, as measured by reduction in phosphatase activity Examples 2 and 3 address the inhibition of H /K ATPase activity m whole cells, as measured by a reduction in acid secretion into the medium Examples 4-9 address in \ιvo models of gastrointestinal inflammatory conditions that are exacerbated by gastπc acidity, generally according to International Publication No WO 96/01624 EXAMPLE 1
Inhibition of ATPase enzymic activity in plasma membrane preparations
The effect of BPI protein products on H7K -ATPase enzymic activity in mouse liver plasma membrane preparations was evaluated Male laboratory mice (Mas musculus) were sacπficed 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 homogemzer and six passes of a teflon pestle in 9 volumes of 0 25 M sucrose with protease inhibitors (2 μg/mL pepstatin A, 2 μg/mL aprotimn and lmM 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 centπfugation 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 ultracentπfuge 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 colonmetπc determination of phosphate release, as follo s Assays were conducted in a 96-well plate Each well contained appioximately 10-15 μL (per lOOμL total volume) microsome suspension in incubation buffer ( 10 mM MES-Tπs, pH 6 5, 25 mM NH4C1) Fiv e μL of one of the following test compounds were added to give the indicated final concentration 1 35 μM rBPI-,,, 0 37 μM omeprazole acid, 0 37 μM omeprazole, 0 25 uM oligomycin, 10 mg/ ml efrapeptin F&G, 0 015 μM sodium azide, 0 076 uM XMP 416 (SEQ ID NO 5), 0 082 μM XMP 391 (SEQ ID NO 4), 0 553 uM XMP 416 and 0 482 μM XMP 391 Each plate contained an Enzyme Blank [test compound and buffer alone, without the ATPase-contaming suspension], a Positive Control [ATPase-containing suspension alone, without test compound added], and a Phosphate Standard [ATPase-containing suspension containing 50 nM phosphate (5 μl of 10 mM NaH,PO4)]
The 96-well plate was incubated 10 min at 21°C (room temperature) The reaction was initiated by adding 50 μl of ATP Stock solution [lOmM MES, 15mM ATP, 15mM MgSO4, 25mM NH4C1, 0 05% (w/v) deoxycholate, adjusted to pH 6 5 with Tns base] to each well The plate was incubated for a total of 15 minutes Plates were centπfuged in a Beckman J-6M centπfuge for 5 minutes at 1200 rpm One hundred μL of each supernatant was transferred to a new 96-well plate One hundred μl of Color Developing Reagent, a combined stop solution and color development reagent, was added [prepared by adding 0.5g Ascorbic acid to 30 ml H2O, followed by adding 5 ml 12% Ammonium Molybdate in 12N H2SO4 and 5 ml of 10% sodium lauryl sulfate, followed by adjusting total volume to 50 ml with H2O] 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 )nm 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 H7K+ ATPase acid secreting activity in cells
These expeπments ere designed to determine the abi tv of RAWT 264 7 cells (a muπne monocytic cell line) to acidify culture medium as they proliferate and metabolize In these experiments, the medium is acidified after 24 hours w ith 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~/IC-ATPase). Compounds that inhibit the acid secretion from mammalian cell lines were evaluated by measuπng 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 Oπon Research Digital Ion analyzer/501 (Beverly, MA) pH meter and Oπon semi-micro microelectrode. Toxic effects of the tested compounds were dissociated from inhibition of acid secretion by measuπng cell proliferation in parallel 96-well microtiter plates with identical medium and identical inhibitor concentrations Proliferation was measured by 3H-thymιdme incorporation over 24 hours using 2 5 x 10 ^RAW 264 7 cells/well Cells were harvested onto glass microfibre filters using an Inotech Cell Harvester (Lansing, MI) and 3H-thymιdme incorporation was quantified on the dπed filters using an Inotech Trace 96 Automatic Filter Counting System radioactivity counter Results of pH and 3H-thymιdιne measurements for XMP 416 are shown in Figure 2 Toxic effects of the peptide at concentrations greater than 50 μg/ml XMP 416 are clearly dissociated from the inhibition of medium acidification at 12.5 μg/ml XMP 416
EXAMPLE 3 Inhibition of H7K+ ATPase acid secreting activity
The inhibition of H /K ATPase activity in gastπc vesicles is measured as follows according to U S Patent No 5,420,135 Lyophi zed gastπc esicles are prepared from pig fundic mucosa according to Keeling et al , Biochem Pharmacol , 34 2967 ( 1985 ) Potassium-stimulated ATPase activity is determined at 37°C in the presence of the following 10 mM PIPES/Tπs buffer, pH 7 0, 2 mM MgSO4, 1 mM KC1, 2 mM Na.ATP and 3-6 μg protem/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 measuπng l4C-amιnopyπne accumulation by parietal cells according to U S Patent No 5,523,303 Gastπc mucosal cells are prepared from rat stomach as follows Wistar rats (130-T60 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 stirπng 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 paπetal cells is determined by their morphology
Acid production by these gastπc parietal cells prepared in this manner is induced by cyclic AMP, histamine (in the presence of 3-ιsobutyl-l - methylxanthine) or carbachol Acid production in the presence and absence of inhibitors is assessed by measuπng the accumulation of i4-C-amιnopyπne The undissociated weak base can penetrate into the acid-containing compartments of the cells In the acidic compartment, the aminopyπne dissociates, after which it can no longer penetrate the membrane because the membrane is impermeable to the dissociated form Thus, the distribution of l4-C-amιnopyπne between the extracellular and intracellular spaces is an indirect quantitativ e index for cellular acid production [Schepp et al , Am J Ph\sιol 259, Gastt ointest Live/ Plnswl 22 G646-654 (1990)] EXAMPLE 4 Inhibition of gastric acid secretion
Inhibition of gastπc secretion is measured according to the method of Shay ligation, Gastroenterologx , 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 gastπc juice is measured The inhibition of gastπc secretion is calculated by compaπng the measured values with those of the control group to which no test compound was administered The EDS0 of the test compound is the dose that inhibits the gastπc 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, 11 761 -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 fio test compound was administered, and the ED^0 of the test compound which inhibits the lesion by 50% is calculated Alternatively, the percentage inhibition of lesion formation may be calculated
EX MPLE 6
Protection against mepirozole-induced duodenal ulcers
The protectiv e effect against the formation of mepirozole-induced duodenal ulcerative lesions is measured as follows Male sprague-Daw ley 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 mepiπzole 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 ED,0 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 indomethac - induced gastπc 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 indomefhacm is subcutaneously administered to cause gastπc lesions The EDS0 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 pπor to carrying out the expeπment, in which the rats are stressed by immersing them in water Thirty minutes pπor to immersing the rats in water, a BPI protein product or omeprazole as a positive control is administered The ED^0 of the test compound which inhibits the lesions by 50% is calculated
EXAMPLE 9 Healing of acetic acid-induced ulcers
The effect on healing of acetic acid-induced ulcerative lesions is evaluated as follows Male sprague-Dawlev rats are starved for 5 hours 20 ul of 30% acetic acid is injected into the submucosal layer of the stomach using a microsyπnge, 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 compared to the control group that received no test compound
Numerous modifications and variations of the above-descπbed invention are expected to occur to those of skill in the art. Accordingly, only such limitations as appear m the appended claims should be placed thereon

Claims

WHAT IS CLAIMED ARE.
1 A method of inhibiting H IK' ATPase activity in a mammal suffeπng from a condition exacerbated by acid secretion involving H7K~ ATPase activity comprising administering to said mammal an amount of a BPI protein product effective to inhibit H7K~ ATPase activity in said mammal.
2 The method of claim 1 wherein the mammal is suffeπng from gastrointestinal ulcer disease
3. The method of claim 1 wherein the mammal is suffeπng from gastrointestinal inflammatory disease or other condition exacerbated by gastnc acidity.
4. The method of claim 1 wherein the BPI protein product is an ammo- terminal fragment of BPI protein having a molecular weight of about 20 kD to 25 kD, or a dimeπc form thereof.
5 The method of claim 1 wherein the BPI protein product is a BPI-deπved peptide
6 The method of claim 5 wherein the BPI-deπved peptide is selected from the group consisting of XMP 391 (SEQ ID NO" 4) and XMP 416 (SEQ ID NO- 5)
7 A method of inhibiting gastπc acid secretion in a mammal suffeπng from a condition exacerbated by gastπc acidity compπsing administeπng to said mammal an amount of a BPI protein product effective to inhibit gastric acid secretion in said mammal
8 The method of claim 7 wherein the mammal is suffering from gastrointestinal ulcer disease
9 The method of claim 7 wherein the mammal is suffeπng from gastrointestinal inflammatory disease or other condition exacerbated by gastπc acidity
10 The method of claim 7 wherein the BPI protein product is an ammo- terminal fragment of BPI protein having a molecular weight of about 20 kD to 25 kD, or a dimeπc form thereof.
11. The method of claim 7 wherein the BPI protein product is a BPI-deπved peptide.
12 The method of claim 1 1 wherein the BPI-deπved peptide is selected from the group consisting of XMP 391 (SEQ ID NO. 4) and XMP.416 (SEQ ID NO. 5).
PCT/US2000/009125 1999-07-12 2000-04-06 Therapeutic uses of bpi protein products for inhibiting h+/k+ atpase activity WO2001003724A1 (en)

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