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WO1993003716A1 - Procede destine a augmenter l'accumulation intracellulaire d'agents anioniques hydrophiles a l'aide de gemfibrizol - Google Patents

Procede destine a augmenter l'accumulation intracellulaire d'agents anioniques hydrophiles a l'aide de gemfibrizol Download PDF

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WO1993003716A1
WO1993003716A1 PCT/US1992/007177 US9207177W WO9303716A1 WO 1993003716 A1 WO1993003716 A1 WO 1993003716A1 US 9207177 W US9207177 W US 9207177W WO 9303716 A1 WO9303716 A1 WO 9303716A1
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cells
therapeutic agent
antibiotic
dimethylphenoxy
structural analog
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Samuel C. Silverstein
Harold C. Neu
Charles Cao
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Columbia University in the City of New York
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    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/429—Thiazoles condensed with heterocyclic ring systems
    • A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
    • 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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/542—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/545—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Lucifer Yellow is transported from the cytoplasmic matrix into endosomes, delivered from endoso e to lysosomes, and also secreted into the extracellular medium.
  • (1,2) Within 30 minutes after the dye is introduced into the cytosol by ATP-mediated permeabilization of the plasma membrane, 80-85% of the dye is secreted by the cells into the medium; the dye remaining in the cells is present within the endocytic compartment and ultimately is transferred to lysosomes.
  • Probenecid and sulfinpyrazone inhibit both intracellular sequestration and secretion of Lucifer Yellow.
  • endogenous substrates for these organic anion transporters are not known, many metabolites and secreted mediators, including bilirubin, glutathione, prostaglandins, and leukotrienes, are substrates for organic anion transporters in various polarized epithelia.
  • This invention provides a method to enhance intracellular accumulation in mammalian cells of a hydrophilic, anionic therapeutic agent which cannot normally accumulate in such cells which comprises contacting the cells with the therapeutic agent and with 5-(2,5-dimethylphenoxy)-2,2- di ethylpentanoic acid, or a structural analog thereof, in an amount effective to block transport of the therapeutic agent from the cells causing intracellular accumulation of the therapeutic agent in the cells.
  • composition comprises a therapeutic agent and 5-(2,5-dimethylphenoxy)-2,2-dimethylpentanoic acid, or a structural analog thereof, and a pharmaceutically acceptable carrier.
  • an improved therapeutic method which comprises administering to a mammal a therapeutic agent and 5-(2,5-dimethylphenoxy)-2,2-dimethyl- pentanoic acid, or a structural analog thereof, in an amount effective to improve the efficacy of the therapeutic agent.
  • a method of treating an intracellular bacterial infection which comprises contacting the infected cell with an effective amount of a therapeutic agent and with an effective amount of 5-(2,5- dimethylphenoxy)-2,2-dimethyl-pentanoic acid, or a structural analog therof, effective to treat an intracellular bacterial infection.
  • FIG. 1 Penicillin G inhibits secretion of Lucifer Yellow from the cytoplasmic matrix of J774 cells.
  • Adherent J774 cells were incubated in D10 containing 5 mM ATP, 0.5 mg/ml Lucifer Yellow, with or without 10 mM penicillin G, for 10 minutes at 37°C. The cells were viewed by fluorescence microscopy at intervals.
  • FIG. 3 Probenecid-sensitive Penicillin G afflux from J774 macrophages. J774 cells were loaded with [ 1 C] penicillin G by ATP-per eabilization. The cells were then incubated in DMEM in the presence (filled circles) or absence (open circles) of 10 mM probenecid. At intervals, the cells were washed, lysed in Triton X-100, and radiolabel was guantitated as described. The results show averages of three separate experiments each done in triplicates, and their standard errors.
  • FIG. 4 Probenecid reversibly increased intracellular retention of norfloxacin. J774 cells were incubated with [ 3 H] norfloxacin in the presence (filled circles) or absence (open circles) of 10 mM probenecid for various times. Some samples in probenecid were washed and reincubated in fresh medium (triangles) . At the end of the incubation, the cells were washed, then lysed and radiolabel counted in a scintillation counter. The results show averages of three or more separate experiments each done in triplicates, and their standard errors.
  • FIG. 6 Intracellular growth of Listeria monocyto ⁇ enes in J774 cells in the presence of 0.2 mM GFZ (open circles) or in the absence of GFZ (filled circles) .
  • the intracellular growth assays were performed as described in Materials and Methods (second series of experiments) which follow. Note that GFZ has no effect on the rate or extent of growth of Listeria monocvto ⁇ enes.
  • FIG. 7 Intracellular growth of Listeria monoc ⁇ to ⁇ enes in J774 cells under three different conditions: 0.2 mM GFZ (open circles), 2 ⁇ g/ml NFX (filled circles) and 0.2 mM GFZ + 2 ⁇ g/ml NFX (open triangles) .
  • the intracellular growth assays were performed as described in Materials and Methods (second series of experiments) which follow. Note that 2 ⁇ g/ml NFX has little inhibitory effect on growth of Listeria while the same concentration of NFX causes marked inhibition of growth when used in combination with 0.2 mM GFZ.
  • J774 cells under three different conditions: 0.2 mM GFZ (open circles), 4 ⁇ g/ml NFX (filled circles) and 0.2 mM GFZ + 4 ⁇ g/ml NFX (open triangles) .
  • the intracellular growth assays were performed as described in Materials and Methods. Note that there was marked inhibition of intracellular growth of Listeria monocvto ⁇ enes in the presence of 4 ⁇ g/ml NFX alone, but this inhibition was markedly potentiated by the addition of 0.2 mM GFZ.
  • FIG. 9 Intracellular growth of Listeria monocvtogenes in J774 cells under three different conditions: 0.2 mM GFZ (open circles), 8 ⁇ g/ml NFX (filled circles) and 0.2 mM GFZ + 8 ⁇ g/ml NFX (open triangles) .
  • the intracellular growth assays were performed as described in Materials and Methods (second series of experiments) which follow. Note that 8 ⁇ g/ml NFX had a bacteriostatic effect while addition of 0.2 mM GFZ plus 8 ⁇ g/ml NFX led to a bactericidal effect on intracellular Listeria monocvtogenes.
  • This invention provides a method to enhance intracellular accumulation in mammalian cells of a hydrophilic, anionic therapeutic agent which cannot normally accumulate in such cells which comprises contacting the cells with the therapeutic agent and with 5-(2,5-dimethylphenoxy)-2,2- dimethylpentanoic acid, or a structural analog thereof, in an amount effective to block transport of the therapeutic agent from the cells causing intracellular accumulation of the therapeutic agent in the cells.
  • the hydrophilic, anionic therapeutic agent which cannot normally accumulate in the cells is an antibiotic.
  • the antibiotic is an antibiotic selected from the group consisting of penicillins, cephalosp ⁇ rins, or guinolones. In the most preferred embodiment, the guinolone is norfloxacin.
  • the contacting of the cell with the therapeutic agent and withthe5-(2,5-dimethylphenoxy)-2,2-dimethylpentanoicacid, or a structural analog thereof, is sequential or simultaneous with respect to each other.
  • an improved therapeutic method which comprises administering to a mammal a therapeutic agent and 5-(2,5-dimethylphenoxy)-2,2-dimethyl ⁇ pentanoic acid, or a structural analog thereof, in an amount effective to improve the efficacy of the therapeutic agent.
  • the mammal may be, but is not limited to a human.
  • the therapeutic agent is an antibiotic, such as an antibiotic selected from the group consisting of penicillins, cephalosporins, or quinolones.
  • the quinolone is norfloxacin.
  • administration of the therapeutic agent and the 5-(2,5-dimethylphenoxy)-2,2- di ethylpentanoic acid, or a structural analog thereof is sequential or simultaneous with respect to each other.
  • a pharmaceutical composition is further provided by this invention wherein the composition comprises a therapeutic agent and 5-(2,5-dimethylphenoxy)-2,2-dimethylpentanoic acid, or a structural analog thereof, and a pharmaceutically acceptable carrier.
  • the therapeutic agent may be, but is not limited to an antibiotic, such as an antibiotic selected from the group consisting of penicillins, cephalosporins, or quinolones.
  • the guinolone is norfloxacin.
  • the therapeutic agent may be, but is not limited to an antibiotic, such as an antibiotic selected from the group consisting of penicillins, cephalosporins, or quinolones.
  • the antibiotic is a quinolone, e.g., norfloxacin.
  • the contacting of the infected cell with the therapeutic agent and with the 5-(2,5-dimethylphenoxy)-2,2- dimethylpentanoic acid, or a structural analog thereof, is sequential or simultaneous, with respect to each other.
  • the intracellular bacterial infection includes, but is not limited to a infection by Listeria, Tuberculosis, Mycoplasma, Legionella, Leprosy, Brucella or Salmonella.
  • the term accumulation is defined as th total concentration of the therapeutic agent within the cell and thus includes that which is located and/or sequestered within intracellular organelles, for example, vacuoles and phagosomes.
  • mammalian cells includes all cell types, including, but not limited to macrophages, nerve cells, neuroblastoma cells, epithelial cells and white blood cells.
  • therapeutic agents useful in the practice of this invention include, but are not limited to antibiotics, such as beta-lactim antibiotics, for example, penicillins, cephalosporins and streptomycin, quinoline antibiotics such as ciprofloxacin, norfloxacin, and hydrophilic, antiviral agents, such as interferon or its hydrophilic, anionic form thereof, and cancer chemotherapeutic agents, for example, doxorubicin hydrochloride, cisplatin and platinum, dia mine [l,l-cyclobutane-decarboxylate(2-)-0,0'], or the anionic, hydrophilic form thereof.
  • antibiotics such as beta-lactim antibiotics, for example, penicillins, cephalosporins and streptomycin
  • quinoline antibiotics such as ciprofloxacin, norfloxacin
  • antiviral agents such as interferon or its hydrophilic, anionic form thereof
  • cancer chemotherapeutic agents for example, doxorubi
  • the method of this invention may be practiced j__ vitro or in vivo. If the method is practiced jn vitro.
  • contacting may be effected by incubating the cells with the agent and with the 5-(2,5-dimethylphenoxy)-2,2-dimethylpentanoic acid, or a structural analog thereof.
  • concentration of the therapeutic agent is effective for its intended purpose and thus, will vary with the cell and purpose of the contacting.
  • Another factor in determining the effective amount of the therapeutic agent the amount or analog of 5-(2,5- dimethylphenoxy)-2,2-dimethylpentanoic acid.
  • the methods of the present invention are intended for the treatment of mammals, including human patients. It also is intended that the therapeutic agent and 5-(2,5-dimethyl phenoxy)-2,2-dimethylpetanoic acid, or a structural analog thereof, be administered as a composition comprising the therapeutic agent and 5-(2,5-dimethyl phenoxy)-2,2- dimethylpetanoic acid (or a structural analog thereof) and a pharmaceutically acceptable carrier.
  • the term "pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water emulsion, and various types of wetting agents.
  • compositions will be in an effective dosage. Administration may be effected continuously or intermittently such that the amount of the composition in the patient is effective.
  • the therapeutic agent may be administered at the same time as, or subsequent to, the administration of 5- (2,5-dimethyl phenoxy)-2,2-dimethylpentanoic acid, or a structural analog therof.
  • J774 cells were grown in spinner culture in Dulbecco's modified Eagle medium (DMEM) containing 10% heat inactivated calf bovine serum, 100 unit/ml penicillin G and 100 ⁇ /ml streptomycin (D10) . Before experiments using penicillin, cells were grown in penicillin-free medium overnight.
  • DMEM Dulbecco's modified Eagle medium
  • D10 100 unit/ml penicillin G and 100 ⁇ /ml streptomycin
  • D10 D10
  • Penicillin G was from New England Nuclear.
  • NJ Dohme Co.
  • Lucifer Yellow CH lithium salt, was from Molecular Probes (Eugene, OR) .
  • Probenecid and sulfinpyrazone were from Sigma (St. Louis, MO).
  • J774 cells were plated at 10 6 cells/well in 24-well tissue culture plates and cultured in D10 at 37°C overnight. The cells were incubated in phosphate buffered saline with divalent cations (PBS) containing 0.5 mg/ml Lucifer Yellow, 5 mM ATP and other compounds, at 37°C as indicated in the text. The wells were washed with cold phosphate buffered saline without divalent cations (PD) 3 times, and plates were immersed successively in three beakers containing 1,000 ml cold PD. The first beaker also contained 0.1% bovine serum albumin.
  • PBS phosphate buffered saline with divalent cations
  • the cells then were lysed in 0.05% Triton X-100, and fluorescence was measured in a fluorescence spectrophotometer using an excitation wavelength of 430 nm and an emission wavelength 540 nm. Total cell protein was measured by a modification of the Lowery method. (.6) Results were expressed as ng Lucifer Yellow/mg protein. r 14 C1-Penicillin G Afflux from J774 Cells: J774 cells were plated at 10 6 cells/well in 24-well tissue culture plates and incubated in D10 with neither penicillin nor streptomycin in the medium at 37 ⁇ C and 5% C0 2 overnight.
  • the cells were incubated in PBS containing 0.5 ⁇ Ci/ml [ 14 C] penicillin G and 5mM ATP for 10 minutes before the loading was terminated by washing the cells with cold PD as described above.
  • DMEM with or without lOmM probenecid was added to the cells and the cells were incubated at 37°C to 5% C0 2 for various time periods. At the end of each time point, the cells were washed with cold PD, the cells were lysed with 0.5% Triton X-100, radioactivity was measured in a scintillation counter, and protein was quantitated as above.
  • J774 cells were prepared as above. The cells were incubated in PBS with 1.5 ⁇ Ci/ml [ 3 H] norfloxacin in the presence or absence of lO M probenecid for various lengths of time, washed and lysed. Cell-associated radioactivity and protein were quantitated as above.
  • L. monocyto ⁇ enes strain 101035 was provided by Dr. D. Portnoy, Univ. of Pennsylvania, Philadelphia, PA. The bacteria were grown in brain heart infusion broth (BHI) and on LB agar (Difco Laboratories, Inc., Detroit, MI) for analysis of MIC and MBC. Bacteria grown in BHI broth were suspended in DME + 5%HICS media (Life Technologies, Inc., Grand Island, NY.) to insert J774 cells. Tissue Culture Cells and Growth Medium
  • J774 macrophage-like cells were grown in DME containing 5% heat inactivated (56 ⁇ C x 30 min) calf serum (HICS) , penicillin (100 U/ml) and streptomycin (10 ⁇ g/ml) .
  • HICS heat inactivated calf serum
  • penicillin 100 U/ml
  • streptomycin 10 ⁇ g/ml
  • 10 6 J774 cells were plated in 60 mm petri dishes (Corning Glass Works, Corning, N.Y.) in DME containing 5% HICS but without antibiotics and incubated overnight at 37°C in the medium.
  • L. monocvtogenes were inoculated into 1 ml of BHI broth and grown overnight in BHI broth to a density of approximately 10 9 bacteria per ml, diluted to a concentration of 2 x- 10 3 bacteria per ml in Brain Heart Infusion Broth, and 0.5 ml aliquots of this suspension were placed in separate tubes.
  • Norfloxacin (NFX) at the indicated concentrations in 0.5 ml BHI broth was added to the tubes containing L. monocyto ⁇ enes. and the bacteria were incubated overnight at 25°C, 30 ⁇ C and 35°C.
  • the MIC of NFX for L. monocvto ⁇ enes was determined by visual inspection (tubes without antibiotic, in which the bacteria grew contained a cloudy suspension) .
  • MCC Minimum Bacteriocidal Concentration
  • J774 cells grown in spinner culture were centrifuged for 5 min. at 1000 RPM.
  • the cells were resuspended in fresh DME + 5% HICS without antibiotics at a concentration of 2xl0 5 .
  • 5 ml of this cell suspension was plated into each 60 mm Petri dish containing multiple 12 x 1 mm round coverslips (Fisher Scientific Co.) the evening before use to allow the cells to form monolayers on the coverslips.
  • the cells were incubated overnight at 37°C.
  • L. monocvto ⁇ enes was incubated overnight in BHI broth to a density of approximately 10 9 /ml in a shaker at 37 ⁇ C. 1 ml of the bacterial suspension was sedimented in a microfuge tube (USA/Scientific Plastics, Ocala, Florida.) for 1 min at 8000 RPM. The supernatant was removed and the pellet was washed once in 1 ml of PBS at pH 7.4.
  • the bacteria were then resuspended in 1 ml fresh DME + 5% HICS, diluted in DME + 5% HICS to a concentration of 2 x 10 s bacteria per ml, and 5 ml of this suspension (10 6 bacteria total) was placed in each 60 mm dish containing a monolayer of J774 cells (infection ratio 1:1). The dishes were incubated at 35°C for 60 minutes. The coverslip was then transferred to a new 60 mm petri dish containing 10 ml of pre warmed DME + 5% HICS and 5 ⁇ g/ml of gentamicin. The cells were incubated in the gentamicin containing medium for 1 hr.
  • the number of bacteria in the J774 cells on each cover slip was determined by depositing each coverslip into 5 ml of sterile distilled water in a 15 ml Falcon tube. The tubes were vortexed for 15 sec. to lyse the infected cells and 10 or 50 ⁇ L of each Listeria- containing solution was plated onto 10 cm dishes containing LB agar. The LB agar plates were incubated at 37°C overnight, the bacterial colonies were counted, and the number of bacteria per cover slip was calculated from the number of colonies on the agar. Every data point represents the average of the number of bacterial colonies recovered from three coverslips.
  • the organic anion transporter of J774 cells limits intracellular accumulation of Lucifer Yellow during ATP permeabilization
  • Extracellular ATP 4 permeabilizes the plasma membrane of J774 cells to small molecules ( ⁇ 900 daltons) such as Lucifer Yellow (7) .
  • small molecules ⁇ 900 daltons
  • Lucifer Yellow (7) .
  • the dye rapidly permeates the cytoplasmic matrix of the cells.
  • Lucifer Yellow enters the cell cytoplasm, most of the dye is secreted into the extracellular medium by probenecid- inhibitable organic anion transporters. (1) Inhibition of the organic anion transporters therefore would be expected to increase the intracellular accumulation of Lucifer Yellow during ATP permeabilization.
  • Penicillin G is a substrate for the organic anion transporter that secretes Lucifer Yellow
  • Penicillin G also reduced the rate of Lucifer Yellow afflux after both compounds were introduced into J774 cells simultaneously by ATP-mediated permeabilization (Figure 2) .
  • Cells were incubated in D10 containing 0.5 mg/ml Lucifer Yellow and 5mM ATP with or without 10 mM penicillin G. The cells were then washed and incubated in fresh medium at 37 ⁇ C. At intervals, the cells were viewed by fluorescence microscopy. When 10 mM penicillin was present in the medium while Lucifer Yellow was introduced into the cells, more Lucifer Yellow was present in the cytoplasmic matrix of the cells at subsequent times.
  • Norfloxacin a quinoline antibiotic with a broad spectrum of antimicrobial activity, is also an organic anion. Unlike Lucifer Yellow and penicillin G, which do not readily enter cells in the absence of ATP 4 , norfloxacin is lipid soluble and crosses cellular membranes readily. Thus, it was possible to assess norfloxacin secretion without permeabilizing the plasma membrane with ATP 4 . J774 cells were incubated in medium containing [ 3 H] norfloxacin in the present of either 10 mM probenecid or 10 mM gluconic acid (control) and intracellular radiolabel content was measured at intervals ( Figure 3) .
  • probenecid enhanced the intracellular accumulation of norfloxacin 3-4 fold compared to cells incubated with gluconic acid. Gluconic acid did not affect the intracellular norfloxacin concentration as compared with the cells incubated with only norfloxacin in PBS (data not shown) . In the absence of probenecid, the intracellular norfloxacin concentration had already reached its plateau after a 10 minute incubation; in the presence of 10 mM probenecid, the intracellular norfloxacin concentration reached plateau at 30 minutes.
  • probenecid was reversible; the J774 cells were incubated with 1.5 ⁇ Ci/ml [ 3 H] norfloxacin and 10 mM probenecid in PBS for 30 minutes; then in some samples the cells were washed and bathed in medium containing only 1.5 ⁇ Ci/ml [ 3 HJ norfloxacin in PBS. After probenecid was removed from the medium, there was a rapid afflux of norfloxacin from the cells followed by a steady state in which no further loss of antibiotic from the cells occurred
  • J774 cells were preloaded with either 10 mM penicillin or 10 mM gluconic acid by ATP-permeabilization, cells then were incubated with [ 3 H] norfloxacin in PBS, and the amount of radiolabel was measured in the cells at intervals as above.
  • penicillin and norfloxacin compete for the same afflux pathway in J774 cells.
  • Mouse macrophages and J774 cells possess organic anion transporters that promote the secretion of water-soluble, membrane-imper eant, anionic fluorescent dyes such as Lucifer Yellow (1) and antimicrobial agents such as penicillin and Norloxacin (NFX) (10) from the cells' cytoplasm into the surrounding medium.
  • Probenecid (PB) and sulfinpyrazone at concentrations of 2-5 mM block secretion of these substances, and thereby enhance their retention or accumulation within J774 cells.
  • PB Probenecid
  • sulfinpyrazone at concentrations of 2-5 mM block secretion of these substances, and thereby enhance their retention or accumulation within J774 cells.
  • Gemfibrozil a fibrin acid used to lower blood lipids, inhibited Lucifer Yellow secretion by J774 cells at a ten-fold lower concentration than PB.
  • GFZ also enhanced the intracellular accumulation of [ 3 H]NFX with an ED50 of 15 ⁇ M, which is 30-fold lower than the ED50 of PB or sulfinpyrazone in enhancing [ 3 H]NFX accumulation by J774 cells.
  • GFZ may be useful in enhancing the intracellular accumulation of fluoroquinolone antibiotics, and other anionic antibiotics and drugs that are membrane- permeant.
  • LM Listeria monocvtogenes grows intracellularly in J774 cells (9) .
  • 4 ⁇ g/ml NFX blocked growth of LM inoculated into brain-heart infusion broth and 8 ⁇ g/ml NFX was bacteriocidal for LM in this medium (MBC) .
  • Gemfibrozil (0.2mM) inhibits organic anion secretion by J774 cells, and thereby enhances intracellular accumulation of NFX four-fold.
  • GFZ (0.2mM) does not inhibit growth of LM in J774 cells.
  • LM-infected J774 cells were incubated in medium containing 2 or 4 ⁇ g/ml NFX and 0.2mM GFZ, intracellular growth of LM was inhibited.
  • Incubation of LM-infected J774 cells in medium containing 8 ⁇ g/ml NFX and 0.2mM GFZ was bacteriocidal for intracellular LM.
  • Listeria monocytogenes is a facultative intracellular pathogen that causes a severe infection of mice.
  • Mice inoculated intravenously with a sublethal dose of Listeria show progressive growth of this organism in their liver and spleen.
  • Mice inoculated with a sublethal dose of Listeria intraperitoneally show growth of Listeria in their peritoneal macrophages, with subsequent spread to liver and spleen.
  • the number of Listeria in each organ or body site can be enumerated by sacrificing the mouse, homogenizing the organ, and plating dilutions of the homogenate on nutrient agar. By counting the number of Listeria clones on the agar, the number of bacteria in the organ or tissue extract can be determined.
  • Penicillin G is hydrophilic and did not attain a measurable concentration in the cytoplasmic matrix of adherent macrophages in these studies. Only when [ 1 C] penicillin G was introduced into the macrophages by ATP-mediated permeabilization, was the effect of probenecid on membrane transport apparent: penicillin was rapidly secreted into the extracellular medium. Penicillin G therefore behaves similarly to Lucifer Yellow in these respects.
  • Norfloxacin is much more lipid soluble than is penicillin G, and accumulates within the cytoplasmic matrix of macrophages when the cells are incubated in medium containing norfloxacin.
  • medium containing [ 3 H] norfloxacin and either probenecid or sulfinpyrazone a marked increase was seen in the intracellular accumulation of radiolabel. Therefore, in the absence of probenecid or sulfinpyrazone, the intracellular accumulation of radiolabeled norfloxacin is limited by concomitant secretion of the drug.
  • organic anion transport blockers can increase the cytosolic concentration of a variety of therapeutic agents that are organic anions and also are lipophilic or otherwise gain access to the cytoplasmic matrix.
  • penicillin G When penicillin G is introduced into the cells by ATP- induced permeabilization, the drug initially must be in the cytoplasmic matrix (except for the small fraction of the drug that is taken up by pinocytosis) . As with Lucifer Yellow, that penicillin G may also be sequestered within endosomes at the same time that it is secreted into the medium.
  • norfloxacin is taken up both by pinocytosis and by diffusion across the plasma membrane and is secreted back into the medium by the organic anion transporters, and presumably is also sequestered within the endocytic compartment.
  • norfloxacin accumulation is enhanced because the drug is not simultaneously secreted from the cells, and thus, accumulation of norfloxacin within endoso es and lysosomes is inhibited at the same time. This also is the case with fura-2 (3) . It is therefore possible that complete inhibition of organic anion transporters paradoxically decreases drug concentration within endocytic compartments and phagosomes at the same time that it increases the total intracellular concentration of a drug.
  • a number of bacterial pathogens including Salmonella, Brucella, Listeria, Legionella, and Mycobacteria, are able to survive in macrophages after being ingested. Many factors influence the ability of antimicrobials to eradicate infections with intracellular pathogens. These include among others the intracellular location of the organism, the concentration of antibiotic in that location, and the sensitivity of the organism to the particular agent under the conditions that prevail there. In some instances, antibiotics that are effective against a pathogen X_ vitro fail to arrest infections with these organism in vivo.
  • a short-half-life organic, anionic transport inhibitor such as Caronamide is a useful adjunct to antibiotic therapy of intracellular pathogens by selectively increasing the antibiotic concentration in phagosomes (8) .

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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

On décrit un procédé thérapeutique perfectionné qui consiste à administrer à un mammifère un agent thérapeutique et l'acide 5-(2,5-diméthylphénoxy)-2,2-diméthyl-pentanoïque, ou un analogue structurel de celui-ci, en une quantité efficace pour augmenter l'efficacité de l'agent thérapeutique. On décrit également un procédé thérapeutique perfectionné qui consiste à administrer à un mammifère un agent thérapeutique et l'acide 5-(2,5-diméthylphénoxy)-2,2-diméthyl-pentanoïque, ou un analogue structurel de celui-ci, en une quantité efficace pour augmenter l'efficacité de l'agent thérapeutique. L'invention se rapporte aussi à une méthode de traitement d'une infection bactérienne intracellulaire, qui consiste à mettre en contact la cellule infectée avec une quantité efficace d'un agent thérapeutique et avec une quantité efficace d'acide 5-(2,5-diméthylphénoxy)-2,2-diméthyl-pentanoïque, ou un analogue structurel de celui-ci, efficace pour traiter une infection bactérienne intracellulaire. On décrit également une composition pharmaceutique, dans laquelle la composition comprend un agent thérapeutique et de l'acide 5-(2,5-diméthylphénoxy)-2,2-diméthyl-pentanoïque, ou un analogue structurel de celui-ci, ainsi qu'un vecteur pharmaceutiquement acceptable.
PCT/US1992/007177 1991-08-19 1992-08-19 Procede destine a augmenter l'accumulation intracellulaire d'agents anioniques hydrophiles a l'aide de gemfibrizol Ceased WO1993003716A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US74696191A 1991-08-19 1991-08-19
US746,961 1991-08-19

Publications (1)

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WO1993003716A1 true WO1993003716A1 (fr) 1993-03-04

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PCT/US1992/007177 Ceased WO1993003716A1 (fr) 1991-08-19 1992-08-19 Procede destine a augmenter l'accumulation intracellulaire d'agents anioniques hydrophiles a l'aide de gemfibrizol

Country Status (2)

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AU (1) AU2546892A (fr)
WO (1) WO1993003716A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0888049A4 (fr) * 1996-02-29 2005-03-16 Univ Columbia Nouvelle activite antimicrobienne du gemfibrozil

Citations (2)

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Publication number Priority date Publication date Assignee Title
US4859703A (en) * 1987-06-15 1989-08-22 Warner-Lambert Company Lipid regulating compositions
US4891220A (en) * 1988-07-14 1990-01-02 Immudyne, Inc. Method and composition for treating hyperlipidemia

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4859703A (en) * 1987-06-15 1989-08-22 Warner-Lambert Company Lipid regulating compositions
US4891220A (en) * 1988-07-14 1990-01-02 Immudyne, Inc. Method and composition for treating hyperlipidemia

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
THE MERCK INDEX, 10ed. 1985, No. 4246. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0888049A4 (fr) * 1996-02-29 2005-03-16 Univ Columbia Nouvelle activite antimicrobienne du gemfibrozil
US7132096B2 (en) 1996-02-29 2006-11-07 The Trustees Of Columbia University In The City Of New York Antimicrobial activity of gemfibrozil

Also Published As

Publication number Publication date
AU2546892A (en) 1993-03-16

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