WO1998050020A1 - Composition and method for treatment of helicobacter pylori infection - Google Patents

Composition and method for treatment of helicobacter pylori infection Download PDF

Info

Publication number
WO1998050020A1
WO1998050020A1 PCT/US1998/009428 US9809428W WO9850020A1 WO 1998050020 A1 WO1998050020 A1 WO 1998050020A1 US 9809428 W US9809428 W US 9809428W WO 9850020 A1 WO9850020 A1 WO 9850020A1
Authority
WO
WIPO (PCT)
Prior art keywords
pylori
helicobacter
composition
urease
approximately
Prior art date
Application number
PCT/US1998/009428
Other languages
French (fr)
Inventor
Stephen Hersey
Otto Froehlich
George Sachs
Original Assignee
Stephen Hersey
Otto Froehlich
George Sachs
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Stephen Hersey, Otto Froehlich, George Sachs filed Critical Stephen Hersey
Publication of WO1998050020A1 publication Critical patent/WO1998050020A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/664—Amides of phosphorus acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/17—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/04—Sulfur, selenium or tellurium; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/42—Phosphorus; Compounds thereof

Definitions

  • the present invention relates to a composition and a method for treating Helicobacter infection in an animal or human, comprising a membrane-permeant urease inhibitor (UI) and a pH adjusting agent (B) sufficient to maintain a low pH in the bacterial microenvironment for a period of time sufficient to be detrimental to the growth of the Helicobacter.
  • UI membrane-permeant urease inhibitor
  • B pH adjusting agent
  • the present invention relates to a and method for treating
  • H. pylori Helicobacter pylori infection in a human, comprising a UI and a
  • UI-B effective to maintain a pH below approximately 6.0 in the bacterial microenvironment, for at least one hour.
  • Helicobacter including, but not limited to, Helicobacter mustelae (H. mustelae), Helicobacter felis (H. felis) and Helicobacter pylori
  • H. pylori are gram-negative bacteria that colonize and grow in the stomach. H. pylori infection is present in about 50% of all humans (Hunt, R.H. Am. J. Med. 100 (suppl 5A):42S-51S, 1996). H. pylori is recognized as a causative agent in the development of chronic gastritis and a critical factor in the recurrence of most cases of gastric and duodenal ulcer disease. (McGowan et al. Gastroenterology 110:926-938, 1996). Further, there is growing evidence that H.
  • pylori is an essential factor in the development of gastric carcinoma, and this bacterium has been categorized as a group I carcinogen by the World Health Organization. Given the prevalence and pathogenicity of H. pylori, there is a need for treatment of infection by this organism.
  • H. pylori is able to survive and grow in the acidic environment of the stomach (Sachs et al. Acid Related Disorders. Sushu Publishing, Palm Beach, FL, 1995). Acid, produced by the parietal cells of the gastric glands of the stomach, acts to kill ingested bacteria, and few bacterial species survive passage through the stomach. Helicobacter is unique in that it is able to colonize and grow in this acidic environment. The metabolic adaptation that allows Helicobacter to colonize and grow in the stomach is its ability to synthesize large amounts of the enzyme, urease (Mobley et al. J. Clin. Microbiol. 26: 831-836, 1988).
  • Urease converts urea to carbon dioxide and ammonia, and the ammonia acts to neutralize the stomach acid by combining with hydrogen ions to form an ammonium ion.
  • the ability to produce ammonia allows the bacterium to maintain a local environment that is more alkaline than the surrounding gastric fluid.
  • the local environment that is influenced by the ammonia produced by urease action is the environment within the periplasmic space.
  • Gram-negative bacteria such as, but not limited to, Helicobacter possess a cell wall (outer membrane) that contains proteins such as porins that function as small molecule transporters, and a cytoplasmic membrane (inner membrane) that represents the major barrier between the cytoplasm and the environment. Between these two membranes lies the periplasmic space which contains proteins and other molecules secreted by the cell.
  • the cytoplasmic membrane of aerobic bacteria contains the oriented redox pumps responsible for generating a potential difference across the cytoplasmic membrane, negative inside, and an inwardly directed pH gradient. The electrochemical gradient of H across the cytoplasmic membrane is used for ATP synthesis and import and export of metabolites.
  • the periplasmic pH in combination with the transmembrane potential, that enables the survival of Helicobacter in the acid environment of the stomach.
  • proteins insert into the cytoplasmic membrane, in part, as a function of a negative interior potential.
  • the potential difference component of the electrochemical gradient for H ions may play a separate role in growth and reproduction of Helicobacter within the acid environment of the stomach.
  • H. pylori In vitro studies demonstrate that the urease activity of H. pylori is essential for their growth and survival in the acid environment of the stomach (Sjostrom et al. Med. Microbiol. 44: 425-433, 1996; Mooney et al. Lancet 335: 1232, 1990). In the absence of urea or urease activity, H. pylori fails to grow at pH 6 or less and is not viable at or below pH 4. In the presence of both urea and urease activity, some growth is sustained at pH 5 and the H. pylori can survive, at least for a short time, at a pH as low as 2-3. In vitro, at neutral pH, approximately pH 6.5 to pH 7.5, H. pylori requires neither urea nor urease activity for its growth (Goldie et al. J. Clin.
  • urease also is found outside the outer membrane (external urease) (Bode et al. Med. Microbiol. Immun. 182:223-242). This is in addition to the cytoplasmic urease found inside the inner membrane (internal urease). It has been thought that the external urease is responsible for generating a cloud of NH3 around the cell and, thereby, lowering extracellular pH in the immediate environment of the H. pylori. As there is no mechanism known for cell secretion of urease, it has been hypothesized that the external urease is derived from lysed bacteria in a process called altruistic death (Phadnis et al.
  • urea protects H. pylori at acid pH, pH 1.0 to pH 2.0, and that AHA removed this protection (Mooney et al., Lancet 335: 1232, 1990; Nakamura et al., J. Jap. Assoc. Infect. Dis. 67:207-1 1, 1993).
  • In vitro studies showed that at neutral pH, pH 6.5 to pH 7.5, AHA inhibited urease activity but did not kill the bacteria.
  • H. pylori associated with ulcer disease use traditional antibiotics such as, but not limited to, amoxicillin, metronidazole and colloidal bismuth subcitrate and inhibitors of gastric acid secretion and combinations thereof (Sachs et al. Acid Related Disorders. Sushu Publishing, Palm Beach, Fl, 1995). These treatments are only partially effective in eliminating H. pylori infections. The reasons for this include, but are not limited to, development of antibiotic resistance by H. pylori, intolerable side-effects, poor patient compliance with a multi-drug regimen that requires up to 11 tablets per day and the expense of the treatment. Moreover, because many antibiotics act by inhibiting bacterial growth, they are ineffective against the population of H.
  • H. pylori that exists in the non-growing survival state induced by low environmental pH. Further, because of the growing recognition of antibiotic abuse in generating drug- resistant strains of all bacteria including H. pylori, there is no medical indication for the use of antibiotics in treating chronic gastritis, a more widespread and potentially more serious problem resulting from H. pylori infection.
  • compositions and methods for the treatment and eradication of Helicobacter infection in an animal or human that is selective for Helicobacter, that affects non-growing as well as growing H. pylori, that does not induce drug-resistant strains of
  • Helicobacter that has minimal side effects, that encourages patient compliance and that can be provided at a reasonable cost.
  • the membrane permeant urease inhibitor (UI)-pH adjusting agent (B) composition (UI-B) of the present invention satisfies the above need by providing a therapeutic composition that is detrimental to the survival of Helicobacter within the gastric mucosa of an animal or a human.
  • This UI-B composition of the present invention inhibits growth and survival of Helicobacter by combining a UI, which inhibits the intracellular urease of the Helicobacter, and a B, which maintains a pH of between approximately 1.5 and 6 in the bacterial microenvironment for at least one hour.
  • UI-B is selective for Helicobacter, affects both non-growing and growing Helicobacter, does not induce drug-resistant strains of Helicobacter, has minimal side effects, encourages patient compliance and is relatively inexpensive to prepare.
  • the present invention comprises one or more pharmaceutically acceptable UIs which exhibit suitable activity at a pH of between approximately 1.5 and approximately 6.0, and one or more pharmaceutically acceptable Bs, which can maintain a pH between approximately 1.5 and approximatley 6.0 for at least 1 hour.
  • UI-B is administered to an animal or human during the interdigestive phase, when the stomach is empty.
  • UI-B is effective as a therapeutic agent for treating a variety of Helicobacter related diseases in an animal and human. Such diseases include, but are not limited to, esophagitis, gastritis, gastric ulcer, duodenal ulcer and gastric cancers. UI-B is effective when used alone and also is effective as an adjunct to other therapeutic agents.
  • Such therapeutic agents include, but are not limited to colloidal bismuth subcitrate and antibiotics including, but not limited to, tetracycline, metronidazole clarithromycin and amoxicillin, H2-histamine receptor antagonists including, but not limited to, cimetidine, ranitidine and famotidine and H -K -ATPase inhibitors including, but not limited to, omeprazole lansoprazole and pantoprazole.
  • antibiotics including, but not limited to, tetracycline, metronidazole clarithromycin and amoxicillin
  • H2-histamine receptor antagonists including, but not limited to, cimetidine, ranitidine and famotidine
  • H -K -ATPase inhibitors including, but not limited to, omeprazole lansoprazole and pantoprazole.
  • Another object of the present invention is to provide a composition and method that is effective for the treatment of H. pylori in a human.
  • Another object of the present invention is to provide a composition and method that is effective in reducing H. pylori infection.
  • Another object of the present invention is to provide a composition and method that is effective in eliminating H. pylori infection.
  • Another object of the present invention is to provide a composition and method that is selective for the treatment of H. pylori infection.
  • Another object of the present invention is to provide a composition and method that is effective in decreasing the pH in the microenvironment of H. pylori.
  • Another object of the present invention is to provide a composition and method that is effective in maintaining a low pH in the microenvironment of H. pylori.
  • Another object of the present invention is to provide a composition and method that is effective in preventing treating gastric cancer in an animal or human.
  • Another object of the present invention is to provide a method that is effecitve in treating or eliminating H. pylori infection without inducing the development of drug-resistant strains of H. pylori.
  • Another object of the present invention is to provide a composition and method that is effective in supporting the normal physiological defense mechanisms against H. pylori infection within the gastrointestinal tract.
  • Another object of the present invention is to provide a composition and method wherein urease inhibitor uptake by H. pylori is maximal.
  • Another object of the present invention is to provide a composition and method that encourages patient compliance.
  • Another object of the present invention is to provide a composition and method that can be prepared in large amounts. Another object of the present invention is to provide a composition and method that can be prepared reproducibly. Another object of the present invention is to provide a composition and method that is relatively inexpensive to prepare.
  • Another object of the present invention is to provide a composition and method that can be stored for a long period of time and remain effective.
  • Another object of the present invention is to provide a composition and method that will have minimal side effects in an animal or human.
  • Fig. 1 A. pH optimum of solubilized H. pylori urease. B. pH optimum for H. pylori urease in the intact cell.
  • FIG. 3. SDS-PAGE analysis of protein synthesis by H. pylori at fixed medium pHs.
  • FIG. 4 SDS-PAGE analysis of the effect of 20 mM urea on H. pylori protein synthesis at fixed medium pHs.
  • the present invention relates to a composition and a method for treating Helicobacter infection in an animal or human, comprising a membrane-permeant urease inhibitor (UI) and a pH adjusting agent (B) sufficient to maintain a low pH in the bacterial microenvironment for a period of time sufficient to be detrimental to the growth of Helicobacter. More particularly, the present invention relates to a and method for treating Helicobacter pylori (H. pylori) infection in a human, comprising a UI and a
  • UI-B effective to maintain a pH below approximately 6.0 in the bacterial microenvironment, for at least one hour.
  • These can be reversible or irreversible urease inhibitors.
  • the urease inhibitors for use in UI-B can readily permeate the outer membrane and the inner membrane of the bacteria.
  • the urease inhibitors are urea analogs including, but not limited to, hydroxyurea, acetohydroxamic acid, and cell permeant phosphoramidates.
  • the urease inhibitors are hydroxyurea and acetohydroxamic acid.
  • any B that can achieve and maintain a pH preferably between approximately pH 1.5 and approximately pH 6.0, more preferably between approximately pH 1.8 and approximately pH 5.0 and most preferably between approximately pH 2.0 and approximately pH 4.0 for at least one hour can be used to practice the present invention.
  • these include, but are not limited to, phosphate buffers, bisulfate buffers and organic acid buffers such as, but not limited to, citrate, succinate, glutarate, tartrate, ascorbate and other mono-, di- and polycarboxylate buffers. More preferably, these include citrate, tartrate and ascorbate buffers. Most, preferably, these include citrate buffers.
  • Administration of an effective amount of a UI, active at a pH between approximately 1.5 and approximately 6.0, in combination with administration of an effective amount of a B, which achieves and maintains a pH of between approximately 1.5 and approximately 6 for at least approximately one hour in the microenvironment of the Helicobacter has both bacteriostatic and bacteriocidal affects on the growth and survival of H. pylori.
  • the intracellular urease not the extracellular urease that is essential for growth and survival of Helicobacter owing to its role in maintaining the pH of the microenvironment. That is, the intracellular urease, active only under acid conditions, is the essential adaptation that enables Helicobacter to colonize, survive and grow in the acid environment of the stomach. Therefore, to be effective, a urease inhibitor must be able to permeate the cell to access the internal urease and, because the internal urease is needed and active only under acid conditions, the acid conditions must be maintained for a period of time sufficient to suppress the growth and survival of the Helicobacter
  • administration of UI-B to an animal or human is a therapeutic treatment that is capable of inhibiting colonization by and growth of Helicobacter in the stomach and of eliminating Helicobacter from the gastrointestinal tract.
  • the UI of UI-B is preferably between approximately 1-200 mg/kg body weight. More referably, the UI is between approximately 5- 100 mg/kg. Most preferably, UI is between approximately 10-80 mg/kg.
  • the B of the UI-B is preferably between approximately 0.001-0.6 mol/kg body weight. More preferably, the B is between approximately 0.01-0.3 mol/kg. Most preferably, the B is between approximately 0.05 and 0.15 mol/kg.
  • the UI-B is provided in pharmaceutically acceptable formulations which may be prepared by various techniques known to those skilled in the art. The UI and the B can be prepared together and administered simultaneously. Alternatively, the UI and the B can be prepared separately and administered sequentially. The formulations are prepared by uniformly and intimately bringing into association the UI and the B with liquid carriers, with finely divided solid carriers, or with both. More preferably the UI-B or the UI and the B are administered in aqueous solutions, in capsules, in tablets, in coated tablets and in sustained release formulations. Most preferably, the UI and the B are administered together as a UI-B solution.
  • the UI-B can be used with any one, all or any combination of additional ingredients, regardless of the carrier/vehicle, used to present it to the Helicobacter. These include, but are not limited to, colloidal bismuth subcitrate, antibiotics, suspending agents, thickening agents, coating agents, flavoring agents and coloring agents.
  • the UI-B may be presented in, for example, sealed ampoules and vials, and may be stored in a powder or a freeze-dried (lyophilized) condition requiring only the addition of a carrier, for example water, immediately prior to use.
  • the UI-B may be presented in carriers including, but not limited to, capsules, tablets, liposomes, microparticles, microemulsions, microspheres nanoparticles, nanoemulsions, nanospheres and with various natural and synthetic neutral or nondegradable polymers which allow for sustained release of the UI-B.
  • the UI-B should be administered during the interdigestive period. That is, at a time the medication is least likely be affected by other ingested materials.
  • the UI-B is administered into an empty stomach. Least preferably, the UI-B is administered just prior to. at the same time as, or shortly after ingestion of food, drink, or medication that itself affects the pH of the stomach.
  • Routes of administration include, but are not limited to, oral, topical and other routes known to those skilled in the art.
  • the volume of the UI-B to be ingested per dose or the volume of fluid to be ingested with the UI-B dose is preferably from approximately 1-500 ml, more preferably from 1-400 ml and most preferably from 1 to 300 ml.
  • the UI-B dose can be administered one or more times per day over one or more days. The dose of UI-B and the number of times it should be administered will depend on the severity of the
  • UI-B Helicobacter infection being treated
  • the particular formulation of the UI-B used and other clinical factors such as the age, size, weight and condition of the animal or human.
  • the dose of UI-B and the number of times it should be administered can be determined by one skilled in the art. It is to be understood that the IU-B of the present invention has application for both veterinary and human use and is effective as a therapeutic agent for treating or eliminating an Helicobacter infection in an animal or a human.
  • Strain ATCC43504 a clinical isolate, of H. pylori is obtained from the ATCC (Bethesda, MD). Bacteria are grown on Trypticase Soy Agar (TSA) plates with 5% sheep blood (Becton Dickinson, Coceysville, MD) in a micro-aerophilic atmosphere (5% O2, 10% CO2, 85% N2) at 37°C for 24 hours prior to harvesting. Bacteria from one plate are harvested in 300 ⁇ l of brain-heart infusion supplemented with 5% fetal calf serum (Difco Labortories, Detroit, MI).
  • Bacteria in suspensions prepared as in Example 1 are suspended in deionized water and lysed by passage through a French pressure cell at 20,000 psi (SLM Aminco, Urbana, IL). The lysates are centrifuged at 12,000xg for 5min to remove particulate material and the supernatant is used as a source of soluble urease. Assay of urease activity.
  • Urease Activity of the bacterial enzyme, Urease, is assayed according to the method of McDonald, et al. (McDonald et al. Enzymologia
  • the vials are stoppered with rubber stoppers fitted with a plastic well containing 50 ⁇ l of 1.0 N NaO ⁇ to trap released CO2.
  • the incubation medium contains l-5mM radioactive urea with a specific activity of lO ⁇ Ci/ ⁇ mol (Amersham, Arlington Heights, IL). Incubations are carried out at 37°C for 5-60 min. The reactions are stopped by injecting lOO ⁇ l of 5N H2SO4 through the rubber stopper into the medium to release any dissolved CO2.
  • the vials are unstoppered and the NaOH in the trapping well is transferred to 7ml of a scintillation cocktail (Hydrocount®, J T Baker Chemicals).
  • Radioactivity is determined by scintillation counting with automatic quench correction (LKB scintillation counter, Wallac, Gaithersburg, MD) and the amount of CO2 released is calculated from the known specific activity of the urea substrate.
  • the activity of urease is expressed as ⁇ mol CO2 released per min per milligram of protein. Protein content of the aliquots is determined by the Lowry method (Lowry et al. Biol. Chem. 1951, 193: 265-275).
  • the solubilized intracellular urease exhibits activity over a broad range of pH with an optimum activity at about pH 7.5. It also is shown that the solubilized intracellular urease is inactive at pH below 5.0.
  • Figure IB shows the pH dependence of urease activity found for the intact bacteria. In this case the urease shows no activity at pH above 6.5, a steep increase of activity in the range of pH 6.5 to pH 5.5, and sustained activity at pH down to 2.5.
  • Protein synthesis is used as a measure of the ability of H pylori to grow under conditions of varying pH Aliquots of bacterial suspension, prepared as in Example 1 , are added to vials containing 3ml of RPMI-1640 medium (Gibco BRL. Gaithersburg, MD) supplemented with
  • Protein synthesis by H pylori is measured as in Example 6 in medium with or without 20mM urea and with pH adjusted over the range
  • EXAMPLE 7 Effect of urease inhibitor on Helicobacter infection in animals.
  • the ferret Mustela putorius
  • H. mustelae a bacterium closely related to H. pylori, and exhibits pathological symptoms of infection , including gastritis, ulcers and gastric cancer, similar to those seen in humans infected by H. pylori.
  • Ferrets are obtained from a colony maintained at the Morehouse School of Medicine (Atlanta, GA) and are tested by serological assay for infection by H. mustelae.
  • the assay is an ELISA test for the presence of antibodies against H. mustelae using a group of antigenic proteins derived from the bacterium. It is found that essentially all of the animals test positive for the presence of H. mustelae.
  • a sample of the animals testing positive are divided into four groups, A-D. All groups are given food and water on a normal schedule. Group A is hand fed, once per day, l-5ml of a pH-neutral buffer solution
  • Group B is hand fed, once per day, l-5ml of an acid buffer (0.1M citric acid solution, pH 2.2, containing 1% sucrose).
  • Group C is hand fed, once per day, 10-80mg/kg of hydroxyurea dissolved in the pH-neutral buffer solution.
  • Group D is hand fed, once per day, 10-80mg/kg of hydroxyurea dissolved in the acid buffer solution.
  • the treatment protocol is maintained for 5-14 days. Three to ten days following cessation of treatment the animals are tested for an active infestation by H. mustelae. An endoscopic biopsy sample is taken under anesthesia and the sample is examined histologically for the presence of H. mustelae. The biopsy samples are scored on the basis of the number of bacteria observed per unit area of tissue. Both the examination and the scoring are done on a blind basis.
  • Volunteer human subjects are selected from a group of clinical patients entering a treatment program that involves an approved therapeutic regimen including the administration of a urease inhibitor. Prior to initiating the treatment regimen the volunteer patients are tested for the presence of H. pylori infection. A serological test for the presence of antibodies against H. pylori is given. It is found that 40-70% of this patient population tests positive for H. pylori. Subjects testing positive are divided into two groups, A and
  • Group A is instructed to follow the recommended therapy regimen without modification.
  • Group B is instructed to modify their regimen with regard to the urease inhibitor by ingesting the urease inhibitor together with
  • both groups are tested for the presence of an active H. pylori infection.
  • the patients discontinue their urease inhibitor therapy for 12-24 hours prior to testing.
  • a urea breath test using a commercial test kit (Meritech) is used to detect the presence of an active infection.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Inorganic Chemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

The present invention relates to a composition and a method for treating Helicobacter pylori (H. pylori) infection in an animal or human, comprising a membrane-permeant urease inhibitor (UI) and a pH adjusting agent (B) sufficient to maintain a low pH in the bacterial microenvironment. More particularly, the present invention relates to a membrane-permeant urease inhibitor (UI) and a pH adjusting agent (B) effective to maintain a pH below approximately 6.0 in the bacterial microenvironment for a period of time sufficient to be detrimental to the growth and survival of the H. pylori. Methods of making UI-B and methods of using UI-B are also disclosed.

Description

COMPOSITION AND METHOD FOR TREATMENT OF HELICOBACTER PYLORI INFECTION
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional
Application No. 60/046,040 filed May 9. 1997.
FIELD OF THE INVENTION
The present invention relates to a composition and a method for treating Helicobacter infection in an animal or human, comprising a membrane-permeant urease inhibitor (UI) and a pH adjusting agent (B) sufficient to maintain a low pH in the bacterial microenvironment for a period of time sufficient to be detrimental to the growth of the Helicobacter.
More particularly, the present invention relates to a and method for treating
Helicobacter pylori (H. pylori) infection in a human, comprising a UI and a
B (UI-B) effective to maintain a pH below approximately 6.0 in the bacterial microenvironment, for at least one hour. Methods of making UI-B and methods of using UI-B are also disclosed.
BACKGROUND OF THE INVENTION
Helicobacter, including, but not limited to, Helicobacter mustelae (H. mustelae), Helicobacter felis (H. felis) and Helicobacter pylori
(H. pylori) are gram-negative bacteria that colonize and grow in the stomach. H. pylori infection is present in about 50% of all humans (Hunt, R.H. Am. J. Med. 100 (suppl 5A):42S-51S, 1996). H. pylori is recognized as a causative agent in the development of chronic gastritis and a critical factor in the recurrence of most cases of gastric and duodenal ulcer disease. (McGowan et al. Gastroenterology 110:926-938, 1996). Further, there is growing evidence that H. pylori is an essential factor in the development of gastric carcinoma, and this bacterium has been categorized as a group I carcinogen by the World Health Organization. Given the prevalence and pathogenicity of H. pylori, there is a need for treatment of infection by this organism.
H. pylori is able to survive and grow in the acidic environment of the stomach (Sachs et al. Acid Related Disorders. Sushu Publishing, Palm Beach, FL, 1995). Acid, produced by the parietal cells of the gastric glands of the stomach, acts to kill ingested bacteria, and few bacterial species survive passage through the stomach. Helicobacter is unique in that it is able to colonize and grow in this acidic environment. The metabolic adaptation that allows Helicobacter to colonize and grow in the stomach is its ability to synthesize large amounts of the enzyme, urease (Mobley et al. J. Clin. Microbiol. 26: 831-836, 1988). Urease converts urea to carbon dioxide and ammonia, and the ammonia acts to neutralize the stomach acid by combining with hydrogen ions to form an ammonium ion. The ability to produce ammonia allows the bacterium to maintain a local environment that is more alkaline than the surrounding gastric fluid.
The local environment that is influenced by the ammonia produced by urease action is the environment within the periplasmic space.
Gram-negative bacteria such as, but not limited to, Helicobacter possess a cell wall (outer membrane) that contains proteins such as porins that function as small molecule transporters, and a cytoplasmic membrane (inner membrane) that represents the major barrier between the cytoplasm and the environment. Between these two membranes lies the periplasmic space which contains proteins and other molecules secreted by the cell. The cytoplasmic membrane of aerobic bacteria contains the oriented redox pumps responsible for generating a potential difference across the cytoplasmic membrane, negative inside, and an inwardly directed pH gradient. The electrochemical gradient of H across the cytoplasmic membrane is used for ATP synthesis and import and export of metabolites. Therefore, it is the periplasmic pH, in combination with the transmembrane potential, that enables the survival of Helicobacter in the acid environment of the stomach. In bacteria, proteins insert into the cytoplasmic membrane, in part, as a function of a negative interior potential. Thus, the potential difference component of the electrochemical gradient for H ions may play a separate role in growth and reproduction of Helicobacter within the acid environment of the stomach.
It is the pH of the bacterial microenvironment, as opposed to the pH of the gastric fluid, that determines bacterial survival. In the context of H. pylori elimination, the importance of regulating the microenvironment pH for H. pylori growth and survival has not been exploited.
In vitro studies demonstrate that the urease activity of H. pylori is essential for their growth and survival in the acid environment of the stomach (Sjostrom et al. Med. Microbiol. 44: 425-433, 1996; Mooney et al. Lancet 335: 1232, 1990). In the absence of urea or urease activity, H. pylori fails to grow at pH 6 or less and is not viable at or below pH 4. In the presence of both urea and urease activity, some growth is sustained at pH 5 and the H. pylori can survive, at least for a short time, at a pH as low as 2-3. In vitro, at neutral pH, approximately pH 6.5 to pH 7.5, H. pylori requires neither urea nor urease activity for its growth (Goldie et al. J. Clin.
Pathol. 44:695-697, 1991) and survival. At alkaline pH, approximately pH > 8, urea is toxic to H. pylori and urease inhibitors actually provide some protection. Accordingly, H. pylori is dependent on urease activity for growth and survival, but only within the narrow range from about pH 6 to about pH 2.
Unlike in other bacteria, in H. pylori, urease also is found outside the outer membrane (external urease) (Bode et al. Med. Microbiol. Immun. 182:223-242). This is in addition to the cytoplasmic urease found inside the inner membrane (internal urease). It has been thought that the external urease is responsible for generating a cloud of NH3 around the cell and, thereby, lowering extracellular pH in the immediate environment of the H. pylori. As there is no mechanism known for cell secretion of urease, it has been hypothesized that the external urease is derived from lysed bacteria in a process called altruistic death (Phadnis et al. Infection and Immunity 64:905-912, 1995). However, this hypothesis does not account for the inactivation of the urease enzyme by stomach acid. We propose that protection of Helicobacter within the stomach results from the activity of intracellular rather than of extracellular urease. The ammonia produced by intracellular urease is highly membrane-permeant and can readily move from the cytoplasm into the periplasmic space where it provides the nearly neutral pH essential for survival of the Helicobacter within the acid environment of the stomach.
The use of the urease inhibitor, acetohydroxamic acid (AHA), to treat H. pylori infection was first proposed by Mooney et al. (Lancet 335: 1232, 1990). In vitro studies also showed that urea protects H. pylori at acid pH, pH 1.0 to pH 2.0, and that AHA removed this protection (Mooney et al., Lancet 335: 1232, 1990; Nakamura et al., J. Jap. Assoc. Infect. Dis. 67:207-1 1, 1993). In vitro studies showed that at neutral pH, pH 6.5 to pH 7.5, AHA inhibited urease activity but did not kill the bacteria. When AHA was tested for its efficacy in eradicating H .pylori in humans, it temporarily inhibited urease activity, as determined by a breath test, but it did not eradicate the bacterial infection (El Nujumi et al. Gut 32:866-870, 1991). Such data led investigators to conclude that AHA would not be able to eradicate an H. pylori infection (Goldie et al. J. Clin. Pathol. 44:695-697, 1991).
Despite the evidence demonstrating the bactericidal effect of urease inhibitors, attempts to utilize urease inhibitors to treat H. pylori infections have failed to eliminate or reduce the bacterial population (El Nujumi et al. Gut 32:866-870, 1991). Neverthless, as urease is necessary for the growth and survival of H. pylori at low gastric pH, this enzyme remains a therapeutic target for the treatment of H. pylori infection. We have discovered that for a urease inhibitor to be effective, it is necessary to ensure that the pH in the bacterial microenvironment is maintained within a range deleterious to H. pylori growth and survival for a sufficient period of time.
Current methods for eradicating H. pylori associated with ulcer disease use traditional antibiotics such as, but not limited to, amoxicillin, metronidazole and colloidal bismuth subcitrate and inhibitors of gastric acid secretion and combinations thereof (Sachs et al. Acid Related Disorders. Sushu Publishing, Palm Beach, Fl, 1995). These treatments are only partially effective in eliminating H. pylori infections. The reasons for this include, but are not limited to, development of antibiotic resistance by H. pylori, intolerable side-effects, poor patient compliance with a multi-drug regimen that requires up to 11 tablets per day and the expense of the treatment. Moreover, because many antibiotics act by inhibiting bacterial growth, they are ineffective against the population of H. pylori that exists in the non-growing survival state induced by low environmental pH. Further, because of the growing recognition of antibiotic abuse in generating drug- resistant strains of all bacteria including H. pylori, there is no medical indication for the use of antibiotics in treating chronic gastritis, a more widespread and potentially more serious problem resulting from H. pylori infection.
Therefore, what is needed is a composition and method for the treatment and eradication of Helicobacter infection in an animal or human that is selective for Helicobacter, that affects non-growing as well as growing H. pylori, that does not induce drug-resistant strains of
Helicobacter, that has minimal side effects, that encourages patient compliance and that can be provided at a reasonable cost.
SUMMARY OF THE INVENTION The membrane permeant urease inhibitor (UI)-pH adjusting agent (B) composition (UI-B) of the present invention satisfies the above need by providing a therapeutic composition that is detrimental to the survival of Helicobacter within the gastric mucosa of an animal or a human. This UI-B composition of the present invention inhibits growth and survival of Helicobacter by combining a UI, which inhibits the intracellular urease of the Helicobacter, and a B, which maintains a pH of between approximately 1.5 and 6 in the bacterial microenvironment for at least one hour. UI-B is selective for Helicobacter, affects both non-growing and growing Helicobacter, does not induce drug-resistant strains of Helicobacter, has minimal side effects, encourages patient compliance and is relatively inexpensive to prepare.
Briefly, the present invention comprises one or more pharmaceutically acceptable UIs which exhibit suitable activity at a pH of between approximately 1.5 and approximately 6.0, and one or more pharmaceutically acceptable Bs, which can maintain a pH between approximately 1.5 and approximatley 6.0 for at least 1 hour. Preferably, UI-B is administered to an animal or human during the interdigestive phase, when the stomach is empty.
UI-B is effective as a therapeutic agent for treating a variety of Helicobacter related diseases in an animal and human. Such diseases include, but are not limited to, esophagitis, gastritis, gastric ulcer, duodenal ulcer and gastric cancers. UI-B is effective when used alone and also is effective as an adjunct to other therapeutic agents. Such therapeutic agents include, but are not limited to colloidal bismuth subcitrate and antibiotics including, but not limited to, tetracycline, metronidazole clarithromycin and amoxicillin, H2-histamine receptor antagonists including, but not limited to, cimetidine, ranitidine and famotidine and H -K -ATPase inhibitors including, but not limited to, omeprazole lansoprazole and pantoprazole.
Accordingly it is an object of the present invention to provide a composition and method that is effective for the treatment of Helicobacter infection in an animal or human.
Another object of the present invention is to provide a composition and method that is effective for the treatment of H. pylori in a human.
Another object of the present invention is to provide a composition and method that is effective in reducing H. pylori infection.
Another object of the present invention is to provide a composition and method that is effective in eliminating H. pylori infection.
Another object of the present invention is to provide a composition and method that is selective for the treatment of H. pylori infection.
Another object of the present invention is to provide a composition and method that is effective in decreasing the pH in the microenvironment of H. pylori.
Another object of the present invention is to provide a composition and method that is effective in maintaining a low pH in the microenvironment of H. pylori.
Another object of the present invention is to provide a composition and method that is effective in inhibiting the intracellular urease within the H. pylori. Another object of the present invention is to provide a composition and method that is effective in preventing and treating esophagitis in an animal or human.
Another object of the present invention is to provide a composition and method that is effective in preventing and treating gastritis in an animal or human. Another object of the present invention is to provide a composition and method that is effective in preventing and treating gastric ulcer disease in an animal or human.
Another object of the present invention is to provide a composition and method that is effective in preventing treating gastric cancer in an animal or human.
Another object of the present invention is to provide a composition and method that is effective in preventing and treating duodenal ulcer disease in an animal or human. Another object of the present invention is to provide a composition and method that is effective as an adjunct to increase the effectiveness of other anti-ulcer therapies in an animal or human.
Another object of the present invention is to provide a method that is effecitve in treating or eliminating H. pylori infection without inducing the development of drug-resistant strains of H. pylori.
Another object of the present invention is to provide a composition and method that is effective as an adjunct to increase the effectiveness of colloidal bismuth subcitrate treatment for H. pylori infection in an animal or human. Another object of the present invention is to provide a composition and method that is effective as an adjunct to increase the effectiveness of antibiotic treatment for H. pylori infection in an animal or human.
Another object of the present invention is to provide a composition and method that is effective in supporting the normal physiological defense mechanisms against H. pylori infection within the gastrointestinal tract.
Another object of the present invention is to provide a composition and method wherein urease inhibitor uptake by H. pylori is maximal.
Another object of the present invention is to provide a composition and method that encourages patient compliance.
Another object of the present invention is to provide a composition and method that can be prepared in large amounts. Another object of the present invention is to provide a composition and method that can be prepared reproducibly. Another object of the present invention is to provide a composition and method that is relatively inexpensive to prepare.
Another object of the present invention is to provide a composition and method that can be stored for a long period of time and remain effective.
Another object of the present invention is to provide a composition and method that will have minimal side effects in an animal or human.
These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiment and the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1. A. pH optimum of solubilized H. pylori urease. B. pH optimum for H. pylori urease in the intact cell.
Fig. 2. Urease activity as a function of urea concentration in intact H. pylori at pH 7.5 and pH 4.5.
FIG. 3. SDS-PAGE analysis of protein synthesis by H. pylori at fixed medium pHs. FIG. 4. SDS-PAGE analysis of the effect of 20 mM urea on H. pylori protein synthesis at fixed medium pHs.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a composition and a method for treating Helicobacter infection in an animal or human, comprising a membrane-permeant urease inhibitor (UI) and a pH adjusting agent (B) sufficient to maintain a low pH in the bacterial microenvironment for a period of time sufficient to be detrimental to the growth of Helicobacter. More particularly, the present invention relates to a and method for treating Helicobacter pylori (H. pylori) infection in a human, comprising a UI and a
B (UI-B) effective to maintain a pH below approximately 6.0 in the bacterial microenvironment, for at least one hour. Methods of making UI-B and methods of using UI-B are also disclosed.
Any pharmaceutically acceptable UI that effectively penetrates the outer membrane (cell wall) and inner membrane of the
Helicobacter and that is active at a pH preferably between approximately pH 1.5 and approximately pH 6.0, more preferably between approximately pH 1.8 and approximately pH 5.0 and most preferably between approximately pH 2.0 and approximately pH 4.0, can be used to practice the present invention. These can be reversible or irreversible urease inhibitors. Preferably, the urease inhibitors for use in UI-B can readily permeate the outer membrane and the inner membrane of the bacteria. More preferably, the urease inhibitors are urea analogs including, but not limited to, hydroxyurea, acetohydroxamic acid, and cell permeant phosphoramidates. Most preferably, the urease inhibitors are hydroxyurea and acetohydroxamic acid.
Any B that can achieve and maintain a pH preferably between approximately pH 1.5 and approximately pH 6.0, more preferably between approximately pH 1.8 and approximately pH 5.0 and most preferably between approximately pH 2.0 and approximately pH 4.0 for at least one hour can be used to practice the present invention. Preferably, these include, but are not limited to, phosphate buffers, bisulfate buffers and organic acid buffers such as, but not limited to, citrate, succinate, glutarate, tartrate, ascorbate and other mono-, di- and polycarboxylate buffers. More preferably, these include citrate, tartrate and ascorbate buffers. Most, preferably, these include citrate buffers.
Administration of an effective amount of a UI, active at a pH between approximately 1.5 and approximately 6.0, in combination with administration of an effective amount of a B, which achieves and maintains a pH of between approximately 1.5 and approximately 6 for at least approximately one hour in the microenvironment of the Helicobacter has both bacteriostatic and bacteriocidal affects on the growth and survival of H. pylori.
Although not wanting to be bound by the following hypothesis, it is believed that it is the intracellular urease, not the extracellular urease that is essential for growth and survival of Helicobacter owing to its role in maintaining the pH of the microenvironment. That is, the intracellular urease, active only under acid conditions, is the essential adaptation that enables Helicobacter to colonize, survive and grow in the acid environment of the stomach. Therefore, to be effective, a urease inhibitor must be able to permeate the cell to access the internal urease and, because the internal urease is needed and active only under acid conditions, the acid conditions must be maintained for a period of time sufficient to suppress the growth and survival of the Helicobacter
Because the UI are not true antibiotics but antimetabolites specific for the urease enzyme, they will not induce development of drug- resistant strains of Helicobacter or other bacteria. It would thus be of great benefit to have a method for the treatment of Helicobacter infection that uses urease inhibitors to significantly reduce the growth and survival of Helicobacter in the stomach.
It is to be understood that administration of UI-B to an animal or human is a therapeutic treatment that is capable of inhibiting colonization by and growth of Helicobacter in the stomach and of eliminating Helicobacter from the gastrointestinal tract.
The UI of UI-B is preferably between approximately 1-200 mg/kg body weight. More referably, the UI is between approximately 5- 100 mg/kg. Most preferably, UI is between approximately 10-80 mg/kg.
The B of the UI-B is preferably between approximately 0.001-0.6 mol/kg body weight. More preferably, the B is between approximately 0.01-0.3 mol/kg. Most preferably, the B is between approximately 0.05 and 0.15 mol/kg. The UI-B is provided in pharmaceutically acceptable formulations which may be prepared by various techniques known to those skilled in the art. The UI and the B can be prepared together and administered simultaneously. Alternatively, the UI and the B can be prepared separately and administered sequentially. The formulations are prepared by uniformly and intimately bringing into association the UI and the B with liquid carriers, with finely divided solid carriers, or with both. More preferably the UI-B or the UI and the B are administered in aqueous solutions, in capsules, in tablets, in coated tablets and in sustained release formulations. Most preferably, the UI and the B are administered together as a UI-B solution.
The UI-B can be used with any one, all or any combination of additional ingredients, regardless of the carrier/vehicle, used to present it to the Helicobacter. These include, but are not limited to, colloidal bismuth subcitrate, antibiotics, suspending agents, thickening agents, coating agents, flavoring agents and coloring agents. The UI-B may be presented in, for example, sealed ampoules and vials, and may be stored in a powder or a freeze-dried (lyophilized) condition requiring only the addition of a carrier, for example water, immediately prior to use. Further, the UI-B may be presented in carriers including, but not limited to, capsules, tablets, liposomes, microparticles, microemulsions, microspheres nanoparticles, nanoemulsions, nanospheres and with various natural and synthetic neutral or nondegradable polymers which allow for sustained release of the UI-B.
The UI-B should be administered during the interdigestive period. That is, at a time the medication is least likely be affected by other ingested materials. Preferably, the UI-B is administered into an empty stomach. Least preferably, the UI-B is administered just prior to. at the same time as, or shortly after ingestion of food, drink, or medication that itself affects the pH of the stomach.
Routes of administration include, but are not limited to, oral, topical and other routes known to those skilled in the art. Depending on the route of administration, the volume of the UI-B to be ingested per dose or the volume of fluid to be ingested with the UI-B dose is preferably from approximately 1-500 ml, more preferably from 1-400 ml and most preferably from 1 to 300 ml. The UI-B dose can be administered one or more times per day over one or more days. The dose of UI-B and the number of times it should be administered will depend on the severity of the
Helicobacter infection being treated, the particular formulation of the UI-B used, and other clinical factors such as the age, size, weight and condition of the animal or human. The dose of UI-B and the number of times it should be administered can be determined by one skilled in the art. It is to be understood that the IU-B of the present invention has application for both veterinary and human use and is effective as a therapeutic agent for treating or eliminating an Helicobacter infection in an animal or a human.
The following examples will serve to further illustrate the present invention without, at the same time, however, constituting any limitation thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims. EXAMPLE 1 Preparation of Helicobacter pylori suspensions.
Strain ATCC43504, a clinical isolate, of H. pylori is obtained from the ATCC (Bethesda, MD). Bacteria are grown on Trypticase Soy Agar (TSA) plates with 5% sheep blood (Becton Dickinson, Coceysville, MD) in a micro-aerophilic atmosphere (5% O2, 10% CO2, 85% N2) at 37°C for 24 hours prior to harvesting. Bacteria from one plate are harvested in 300 μl of brain-heart infusion supplemented with 5% fetal calf serum (Difco Labortories, Detroit, MI).
EXAMPLE 2 Preparation of soluble urease from H. pylori
Bacteria in suspensions prepared as in Example 1 are suspended in deionized water and lysed by passage through a French pressure cell at 20,000 psi (SLM Aminco, Urbana, IL). The lysates are centrifuged at 12,000xg for 5min to remove particulate material and the supernatant is used as a source of soluble urease. Assay of urease activity.
Activity of the bacterial enzyme, Urease, is assayed according to the method of McDonald, et al. (McDonald et al. Enzymologia
14 1972, 42: 1-9) as the release of radioactive CO2 from C-urea. Aliquots
(l-10μl) of bacterial suspensions prepared as in Example 1 , or of soluble urease prepared as in Example 2, are added to 3ml of incubation medium in
10ml plastic vials. The vials are stoppered with rubber stoppers fitted with a plastic well containing 50μl of 1.0 N NaOΗ to trap released CO2. The incubation medium contains l-5mM radioactive urea with a specific activity of lOμCi/μmol (Amersham, Arlington Heights, IL). Incubations are carried out at 37°C for 5-60 min. The reactions are stopped by injecting lOOμl of 5N H2SO4 through the rubber stopper into the medium to release any dissolved CO2. The vials are unstoppered and the NaOH in the trapping well is transferred to 7ml of a scintillation cocktail (Hydrocount®, J T Baker Chemicals). Radioactivity is determined by scintillation counting with automatic quench correction (LKB scintillation counter, Wallac, Gaithersburg, MD) and the amount of CO2 released is calculated from the known specific activity of the urea substrate. The activity of urease is expressed as μmol CO2 released per min per milligram of protein. Protein content of the aliquots is determined by the Lowry method (Lowry et al. Biol. Chem. 1951, 193: 265-275).
EXAMPLE 3 Effect of external pH on urease activity.
Aliquots of bacterial suspensions or soluble urease are assayed for urease activity as in Example 3 under conditions of different medium pH within the range of 2.5 to 8.5. The medium is strongly buffered, 0.1M phosphate buffer, to prevent any change of pH over the course of the incubation
As shown in Figure 1A, the solubilized intracellular urease exhibits activity over a broad range of pH with an optimum activity at about pH 7.5. It also is shown that the solubilized intracellular urease is inactive at pH below 5.0. Figure IB shows the pH dependence of urease activity found for the intact bacteria. In this case the urease shows no activity at pH above 6.5, a steep increase of activity in the range of pH 6.5 to pH 5.5, and sustained activity at pH down to 2.5.
These results demonstrate that the urease of intact H. pylori is active only at acid pH, i.e., pH below 6.0, and that the urease retains activity at a pH below that required to inactivate the solubilized intracellular enzyme.
EXAMPLE 4 Effect of urea concentration on urease activity.
Aliquots of bacterial suspensions are assayed for urease activity as in Example 3 in medium buffered to pH 7.5 (O.IM phosphate) or pH 4.5 (0.1M citrate) containing varying concentrations of urea over the range of lOμM to 250mM. As shown in Figure 2, the urease activity at pH 4.5 exhibits an apparent Km for urea of about 0.5mM. At a pH of 7.5 the urease activity exhibits an apparent Km for urea in excess of 250mM. The apparent Km for urea of the solubilized intracellular urease at pH 7.5 is about 0.4mM, i.e., similar to that of the urease of intact bacteria at pH 4.5. These results, together with those of Example 4, demonstrate that most, if not all, of the urease of intact bacteria is located withm the cell cytoplasm where it is not exposed directly to the pH of the medium
EXAMPLE 5
Effect ofpH on protein synthesis by H pylori
Protein synthesis is used as a measure of the ability of H pylori to grow under conditions of varying pH Aliquots of bacterial suspension, prepared as in Example 1 , are added to vials containing 3ml of RPMI-1640 medium (Gibco BRL. Gaithersburg, MD) supplemented with
2mM glutamme, 1% non-essential amino acids (100X), ImM pyruvate, 0 1M HEPES buffer, and lOOμCi of 35S-methιonme (Amersham) The medium is adjusted to various pH ovei the range of 3 0 to 9 0 The bacteria are incubated at 37°C for 4h in a micioaerophilhc atmosphere (5% O2, 10% CO2, 85% N2) The bacterial suspensions aie centrifuged at 600xg for
25mιn and the resulting bacterial pellet is resuspended in 400μl of a polyacrylamide gel loading buffer ( 2% sodium dodecyl sulfate, 60mM Tπs buffer, pH 6 8, 10% sucrose, and 005% bromophenol blue tracking dye) The bacteria are lysed by boiling for 5 mm An aliquot, containing about 100μg protein is loaded into wells of a 10%, precast polyacrylamide slab gel (Bio-Rad, Hercules, CA) and the proteins separated by electrophoresis The gels are scanned with a phosphor-imaging system to detect and quantitate the radioactivity lncorpoiated into the proteins (Ambis, Inc , San Diego, CA) As shown in Figure 3, synthesis of protein by H pylori is optimal at about pH 7 0, declines below about pH 6 5, and ceases below about pH 5 5 These results demonstrate that H pylori does not grow below about pH 5 0
EXAMPLE 6
Effect of urea on protein synthesis by H pylori
Protein synthesis by H pylori is measured as in Example 6 in medium with or without 20mM urea and with pH adjusted over the range
5 0 to 7 0 Figure 4 shows that protein synthesis occurs at pH 5 0 in the presence of urea but not in its absence These results demonstrate that protein synthesis, hence growth, by H. pylori below about pH 6.0 requires activity of internal urease.
EXAMPLE 7 Effect of urease inhibitor on Helicobacter infection in animals.
The ferret ( Mustela putorius ) is used as a model animal for testing the effects of urease inhibitor on Helicobacter infections. This animal is the natural host for H. mustelae, a bacterium closely related to H. pylori, and exhibits pathological symptoms of infection , including gastritis, ulcers and gastric cancer, similar to those seen in humans infected by H. pylori.
Ferrets are obtained from a colony maintained at the Morehouse School of Medicine (Atlanta, GA) and are tested by serological assay for infection by H. mustelae. The assay is an ELISA test for the presence of antibodies against H. mustelae using a group of antigenic proteins derived from the bacterium. It is found that essentially all of the animals test positive for the presence of H. mustelae.
A sample of the animals testing positive are divided into four groups, A-D. All groups are given food and water on a normal schedule. Group A is hand fed, once per day, l-5ml of a pH-neutral buffer solution
(0.1M Na citrate, pH 7.2, plus 1% sucrose). Group B is hand fed, once per day, l-5ml of an acid buffer (0.1M citric acid solution, pH 2.2, containing 1% sucrose). Group C is hand fed, once per day, 10-80mg/kg of hydroxyurea dissolved in the pH-neutral buffer solution. Group D is hand fed, once per day, 10-80mg/kg of hydroxyurea dissolved in the acid buffer solution.
The treatment protocol is maintained for 5-14 days. Three to ten days following cessation of treatment the animals are tested for an active infestation by H. mustelae. An endoscopic biopsy sample is taken under anesthesia and the sample is examined histologically for the presence of H. mustelae. The biopsy samples are scored on the basis of the number of bacteria observed per unit area of tissue. Both the examination and the scoring are done on a blind basis.
Animals in Groups A,B&C will be positive for H. mustelae. Animals in Group D will be negative for H. mustelae EXAMPLE 8 Effect of urease inhibitor on H. pylori infection in human.
Volunteer human subjects are selected from a group of clinical patients entering a treatment program that involves an approved therapeutic regimen including the administration of a urease inhibitor. Prior to initiating the treatment regimen the volunteer patients are tested for the presence of H. pylori infection. A serological test for the presence of antibodies against H. pylori is given. It is found that 40-70% of this patient population tests positive for H. pylori. Subjects testing positive are divided into two groups, A and
B. Group A is instructed to follow the recommended therapy regimen without modification. Group B is instructed to modify their regimen with regard to the urease inhibitor by ingesting the urease inhibitor together with
200ml of an acid-buffered solution (0.1M citric acid, pH 2.2, containing 1% sucrose) after a 2-3 hour fast.
After 5-14 days of treatment both groups are tested for the presence of an active H. pylori infection. The patients discontinue their urease inhibitor therapy for 12-24 hours prior to testing. A urea breath test using a commercial test kit (Meritech) is used to detect the presence of an active infection.
Patients receiving the urease inibitor alone will test positive for H. pylori. Patients receiving the urease inhibitor in combination with the acid buffered solution will test negative for H. pylori
While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Claims

CLAIMS We claim:
1. A composition for the treatment of Helicobacter infection in an animal or human, comprising: a. an effective amount of a pharmaceutically acceptable cell permeating urease inhibitor and b. an effective amount of a pharmaceutically acceptable pH adjusting agent that achieves and maintains a pH in the Helicobacter microenvironment of between approximately 1.5 and approximately 6.0 for at least one hour.
2. The composition of claim 1 , wherein the Helicobacter is selected from the group consisting of Helicobacter mustelae (H. mustelae), Helicobacter felis (H. felis) and Helicobacter pylori (H. pylori).
3. The composition of claim 2, wherein the Helicobacter is H. pylori.
4. The composition of claim 1, wherein the urease inhibitor is a urea analog.
5. The composition of claim 4, wherein the urea analog is selected from the group consisting of hydoxyurea, acethydroxamic acid and cell permeant phosphoramidates.
6. The composition of claim 5, wherein the urea analog is hydoxyurea.
7. The composition of claim 1, wherein the pH adjusting agent is selected from the group consisting of phosphate buffer, bisulfate buffer and organic acid buffers.
8. The composition of claim 7, wherein the pH adjusting agent is an organic acid buffer.
9. The composition of claim 8, wherein the organic acid buffer is selected from the group consisting of citrate buffer, succinate buffer, glutarate buffer, tartrate buffer and ascorbate buffer
10. The composition of claim 9, wherein the organic acid buffer is citrate buffer.
11. The composition of claim 1 , wherein the pH is between approximately 1.8 and 5.0
12. The composition of claim 1 1 , wherein the pH is between approximately 2.0 and 4.0.
13. A method for treaing Helicobacter infection in an animal or human in need of such treatment comprising administering to the animal or human: a. an effective amount of a pharmaceutically acceptable cell permeating urease inhibitor and b. an effective amount of a pharmaceutically acceptable pH adjusting agent that achieves and maintains a pH in the Helicobacter microenvironment of between approximately 1.5 and approximately 6.0 for at least one hour.
14. The method of claim 13, wherein the Helicobacter is selected from the group consisting of Helicobacter mustelae (H. mustelae),
Helicobacter felis (H. felis) and Helicobacter pylori (H. pylori).
15. The method of claim 13, wherein the urease inhibitor is a urea analog.
16. The method of claim 13, wherein the pH adjusting agent is selected from the group consisting of phosphate buffer, bisulfate buffers and organic acid bufffers.
17. A method for treating H. pylori infection in a human in need of such treatment comprising administering to the human: a. an effective amount of a pharmaceutically acceptable cell permeating urease inhibitor and b. an effective amount of a pharmaceutically acceptable pH adjusting agent that achieves and maintains a pH in the H. pylori microenvironment of between approximately 2.0 and approximately 4.0 for at least one hour.
PCT/US1998/009428 1997-05-09 1998-05-08 Composition and method for treatment of helicobacter pylori infection WO1998050020A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US4604097P 1997-05-09 1997-05-09
US60/046,040 1997-05-09

Publications (1)

Publication Number Publication Date
WO1998050020A1 true WO1998050020A1 (en) 1998-11-12

Family

ID=21941254

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/009428 WO1998050020A1 (en) 1997-05-09 1998-05-08 Composition and method for treatment of helicobacter pylori infection

Country Status (1)

Country Link
WO (1) WO1998050020A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019186570A1 (en) 2018-03-29 2019-10-03 Yeda Research And Development Co. Ltd. Methods of disrupting a biofilm and/or preventing formation of same

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Chemical Abstracts Service (C A S); 1 January 1900 (1900-01-01), XP002913760, Database accession no. 124-339881 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019186570A1 (en) 2018-03-29 2019-10-03 Yeda Research And Development Co. Ltd. Methods of disrupting a biofilm and/or preventing formation of same

Similar Documents

Publication Publication Date Title
US5334582A (en) Pharmaceutical bacteriocin compositions and methods for using the same
AU782624B2 (en) Methods and compositions for inhibiting adhesion by microorganisms
Megraud et al. Activity of lansoprazole against Helicobacter pylori
KR20050043763A (en) Compositions for the treatment of infectious diseases
CA2241464C (en) Nisin in combination with glycerol monolaurate active against helicobacter
CN1302183A (en) Xylitol compositions for treating upper respiratory conditions
ES2339003T3 (en) RIFALAZIL TO TREAT INFECTIONS BY CLOSTRIDIUM DIFFICILE.
Stables et al. Gastric anti‐secretory, mucosal protective, anti‐pepsin and anti‐Helicobacter properties of ranitidine bismuth citrate
WO2000050069A1 (en) A means for the prophylactic and therapeutic treatment of streptococcal infections
US8765123B1 (en) Compositions and methods for the treatment and prevention of infections caused by Staphylococcus aureus bacteria
CN105031628A (en) Compositions and methods for the treatment or the prevention oral infections by e. coli
US5560912A (en) Method for inhibiting growth of helicobacter pylori
AU672384B2 (en) Pharmaceutical bacteriocin compositions
CN105263489B (en) Antimicrobial compositions and methods of making the same
US5472695A (en) Therapeutic application of a thyme extract and in - vitro methods for inhibiting the growth and urease activity of helicobacter pylori
PT655249E (en) MOENOMYCIN AS A MEDICATION FOR THE TREATMENT OF GASTRIC ULCERS
WO1998050020A1 (en) Composition and method for treatment of helicobacter pylori infection
Yoshiyama et al. A novel in vitro effect of the mucosal protective agent sofalcone–inhibition of chemotactic motility in Helicobacter pylori
JPH08502725A (en) Use of Vacuolar H +++ ATPase inhibitors
US20040204493A1 (en) Carbonic anhydrase inhibitors as drugs to eradicate Helicobacter pylori in the mammalian, including human, stomach
WO1997025032A2 (en) Monoglyceride compositions and their use against helicobacter
EP0577481A1 (en) New therapeutic application of a Thyme extract and in-vitro methods for inhibiting the growth and urease activity of Helicobacter pylori
Figura et al. Activity of omeprazole on Helicobacter pylori and relation to toxicity of strains.
US6380217B1 (en) Methods for controlling GGT-positive bacteria
RU2239451C2 (en) Method for therapeutic or prophylactic treatment of streptoococcus infection (variants) and composition designated for applying in it (variants), lysine enzyme associated with phage of streptococcus group c as active component of pharmaceutical composition

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA