WO2001003724A1 - Utilisations therapeutiques de produits de proteine bpi pour inhiber l'activite de la h+/k+ atpase - Google Patents

Utilisations therapeutiques de produits de proteine bpi pour inhiber l'activite de la h+/k+ atpase 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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English (en)
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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/fr

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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

La présente invention concerne de nouvelles utilisations thérapeutiques de produits de protéine BPI impliquant l'inhibition de l'activité de la H+/K+ ATPase, y compris l'inhibition de la sécrétion d'acide gastrique.
PCT/US2000/009125 1999-07-12 2000-04-06 Utilisations therapeutiques de produits de proteine bpi pour inhiber l'activite de la h+/k+ atpase WO2001003724A1 (fr)

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US10450348B2 (en) 2013-11-06 2019-10-22 Norwegian University Of Science And Technology Antimicrobial agents and their use in therapy
US10517923B2 (en) 2013-11-06 2019-12-31 Norwegian University Of Science And Technology Immunosuppressive agents and their use in therapy
US11246907B2 (en) 2013-11-06 2022-02-15 Norwegian University Of Science And Technology Immunosuppressive agents and their use in therapy
US11337427B2 (en) 2013-11-06 2022-05-24 Norwegian University Of Science And Technology Antimicrobial agents and their use in therapy

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