METHODS FOR PREVENTING OR TREATING INFECTIOUS DISEASES CAUSED BY EXTRACELLULAR MICROORGANISMS, INCLUDING ANTIMICROBIAL-RESISTANT STRAINS THEREOF, USING GALLIUM COMPOUNDS

Abstract

The present invention relates to methods for preventing or treating infectious diseases caused by extracellular microorganisms, such as bacteria and fungi, by systemically administering to a patient a compound containing gallium. The extracellular microorganisms targeted by the present methods include methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE), E. coli O157:H7, fluoroquinolone-resistant Salmonella typhi, and the like. Furthermore, in the present methods, gallium compounds can be co-administered with one or more conventional antimicrobial agents to treat infectious diseases with reduced risks of creating multi-drug resistant pathogens. The methods of the present invention is also applicable to those microorganisms, such as ulcer-causing Helicobacter pylori, complete eradication of which so far has been difficult to achieve.

Claims

1. A method for treating an infectious disease caused by methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), E. coli O157:H7, fluoroquinolone-resistant Salmonella typhi, ceftazidime-resistant Klebsiella pneumoniae, or fluoroquinolone-resistant Neisseria gonorrhoeae in the bloodstream of a subject, the method comprising: administering to the subject a therapeutically effective amount of a gallium compound selected from the group consisting of gallium nitrate, gallium maltolate, gallium citrate, gallium phosphate, gallium chloride, gallium fluoride, gallium carbonate, gallium formate, gallium acetate, gallium sulfate, gallium tartrate, gallium oxalate, and gallium oxide, wherein said therapeutically effective amount is sufficient to reduce the number of, to suppress the growth of, or to kill the methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), E. coli O157:H7, fluoroquinolone-resistant Salmonella typhi, ceftazidime-resistant Klebsiella pneumoniae, or fluoroquinolone-resistant Neisseria gonorrhoeae in the bloodstream of the subject.

2. The method of claim 1, further comprising co-administering a therapeutically effective amount of at least one additional antimicrobial agent.

3. The method of claim 2, wherein the additional antimicrobial agent is vancomycin and/or linezolid.

4. The method of claim 1, wherein the gallium compound is administered orally, intravenously, intramuscularly, subcutaneously, intraperitoneally, or by suppositories.

5. A method for treating an infection in the bloodstream of a subject, the infection caused by methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), E. coli O157:H7, fluoroquinolone-resistant Salmonella typhi, ceftazidime-resistant Klebsiella pneumoniae, or fluoroquinolone-resistant Neisseria gonorrhoeae, said method comprising: administering to the subject a therapeutically effective amount of a gallium compound selected from the group consisting of gallium nitrate, gallium maltolate, gallium citrate, gallium phosphate, gallium chloride, gallium fluoride, gallium carbonate, gallium formate, gallium acetate, gallium sulfate, gallium tartrate, gallium oxalate, and gallium oxide, wherein said therapeutically effective amount is sufficient to reduce the number of, to suppress the growth of, or to kill the methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), E. coli O157:H7, fluoroquinolone-resistant Salmonella typhi, ceftazidime-resistant Klebsiella pneumoniae, or fluoroquinolone-resistant Neisseria gonorrhoeae in the bloodstream of the subject.

6. The method of claim 5, further comprising co-administering a therapeutically effective amount of at least one additional antimicrobial agent.

7. The method of claim 6, wherein the additional antimicrobial agent is vancomycin and/or linezolid.

8. The method of claim 5, wherein the gallium compound is administered orally, intravenously, intramuscularly, subcutaneously, intraperitoneally, or by suppositories.

Description

5. EXAMPLES

[0039] The following examples are provided to further illustrate the current invention but are not intended to in any way limit the scope of the current invention.

5.1. In Vitro Study: Susceptibility of Microorganisms to Gallium

Example 1

[0040] Susceptibility of various microorganisms to gallium was tested by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for each microorganism using gallium nitrate. In general, MIC is determined by (i) mixing a series of broths, each containing a standard number of microorganisms, with serially diluted solutions of the gallium compound; and (ii) determining the MIC, after incubation, that is the lowest concentration of the gallium compound that inhibits the growth of the microorganism. The lower the MIC, the more susceptible the organism is. The MBC is determined by subculturing an aliquot of each sample from the MIC test on an appropriate agar plate containing no gallium compound. After incubation, the MBC is determined to be the lowest concentration of the gallium compound at which no growth is observed.

[0041] Specifically, in the present experiment, two grams of gallium nitrate powder were dissolved in 10 ml of filter-sterilized deionized water and the resulting 20% (w/v) (i.e., 200 mg/ml) solution was once again filter-sterilized. Two-fold serial dilutions were prepared in sterile deionized water down to 0.156% (i.e., 1.56 mg/ml) for the tests for most of the organisms, except for the test for Candida albicans, in which 10%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005% and 0.001% of gallium nitrate solutions were prepared.

[0042] Table 1 shows the list of microorganisms tested for MIC and MBC. All organisms were obtained from the American Type Culture Collection (ATCC), Manassas, Va. Each microorganism was picked from the seed culture (see Table 1) and inoculated in an appropriate type of broth to obtain a 0.5 McFarland turbidity standard. The standard suspension of the microorganism was then diluted to 1:100 with the broth and used for the tests.

TABLE-US-00001 TABLE 1 TEST ORGANISM ATCC # SEED CULTURE Candida albicans 10231 On Sabouraud dextrose agar, at 25-30 C. for 24-48 hours Methicillin-resistant 33592 On tryptic soy agar with Staphylococcus aureus Staphylococcus aureus 5% (MRSA) sheep blood (BAP), at 35- Vancomycin-resistant 51575 37 C. for 24-48 hours Enterococcus faecalis (VRE) Escherichia coli O157:H7 35150 Salmonella typhi 6539 Campylobacter jejuni 29428 On Brucella agar with 5% sheep blood, at 35-37 C. for 48 hours under microaerophilic conditions .sup.aAntibiotics resistance of the organism was confirmed by CLSI (Clinical Laboratory Standards Institute) Oxacillin disk-diffusion test. The zone of inhibition was 6 mm (CLSI Oxacillin resistant range: 10 mm). .sup.bAntibiotics resistance of the organism was confirmed by CLSI Vancomycin disk-diffusion test. The zone of inhibition was 10 mm (CLSI Vancomycin resistant range: 14 mm).

[0043] Each microorganism was tested in duplicate by either a microdilution broth method in 96-well plates (i.e., 0.1 ml of the gallium nitrate solution mixed with 0.1 ml of the microorganism suspension) or a macrodilution broth method in test tubes (i.e., 1 ml of the gallium nitrate solution mixed with 1 ml of the microorganism suspension) as follows:

[0044] Microdilution broth method: Candida albicans; Escherichia coli O157:H7; and Campylobacter jejuni.

[0045] Macrodilution broth method: Methicillin-resistant Staphylococcus aureus (MRSA); Vancomycin-resistant Enterococcus faecalis (VRE); and Salmonella typhi.

[0046] The growth of the microorganisms were determined by visual observation of turbidity in the samples.

[0047] The following controls were incubated together with the test samples:

[0048] Viability control: A mixture of equal volumes of deionized water and an appropriate broth inoculated with a test microorganism but without gallium nitrate; and

[0049] Sterility control: A mixture of equal volumes of deionized water and an appropriate broth without either microorganisms or gallium nitrate.

[0050] Purity of each microorganism was confirmed by streaking an appropriately diluted suspension of the microorganism onto an appropriate agar plate to obtain isolated colonies and observing colony morphology.

[0051] The concentrations of microorganisms in the suspension used in MIC test were determined by inoculating serial dilutions of the suspensions onto appropriate agar plates and counting the number of colonies.

[0052] To determine MBC, 10 l of each sample used in MIC were inoculated onto an appropriate agar plate and incubated. The lowest concentration of the gallium nitrate that showed no growth was determined to be the MBC.

[0053] The results are shown in Table 2 below.

TABLE-US-00002 TABLE 2 Final Conc Broth Agar Plate Incubation of Organism MIC MBC (MIC) (MBC) Condition (CFU/ml) (mg/ml) (mg/ml) Candida Sabouraud Sabouraud At 27 for 9.75 10.sup.5 10 >100 albicans Dextrose dextrose 48 hours agar Methicillin- Muller Tryptic soy At 36 for 5.2 10.sup.5 ND* 12.5 resistant Hinton agar with 48 hours Staphylococcus 5% sheep aureus blood (MRSA) Vancomycin- Muller Tryptic soy At 36 for 3.9 10.sup.5 ND 25 resistant Hinton agar with 48 hours Enterococcus 5% sheep faecalis (VRE) blood Escherichia coli Muller Tryptic soy At 36 for 1.38 10.sup.6 ND 6.25 O157:H7 Hinton agar with 48 hours 5% sheep blood Salmonella typhi Muller Tryptic soy At 36 for 8.3 10.sup.5 ND 6.25 Hinton agar with 48 hours 5% sheep blood Campylobacter Muller Tryptic soy At 36 for 4.9 10.sup.5 ND <0.78 jejuni Hinton agar with 48 hours 5% sheep blood *ND: Not determined due to non-specific turbidity caused by the precipitation of gallium nitrate at some dilutions.

5.2. In Vivo Study: Effect of Gallium Nitrate in Animal Models

Example 2

[0054] Methicillin-Resistant Staphylococcus aureus (MRSA)

[0055] Adult BALBc mice are inoculated with 110.sup.6 CFU/mouse of Staphylococcus aureus-MRSA strain (e.g., ATCC 33592) by intraperitoneal injection. Following bacterial injections (approximately 8 hours post-inoculation), each mouse receives a single intravenous injection of one of the following: 0.9% saline (control), 30 mg/kg, 45 mg/kg, or 60 mg/kg of gallium nitrate, 200 mg/kg of vancomycin, or 45 mg/kg of gallium nitrate and 200 mg/kg of vancomycin, all in 0.9% saline. Initially, there are 5 mice in each of the six groups. Following inoculation, the mice are monitored twice daily for morbidity. Body temperature is obtained twice daily and a mouse whose body temperature decreases by 4 C. or greater will be considered moribund and euthanized. Body weights are taken once daily for the duration of the study. On Day 5, all remaining animals are euthanized. Spleen, lymph nodes and kidneys are collected, homogenized in sterile PBS and serially diluted for bacterial quantitation.

Example 3

[0056] Vancomycin-Resistant Enterococcus faecalis (VRE)

[0057] Adult CF1 mice are caged individually and total counts of native enterococci and possible VRE in colony forming unit (CFU) per gram of feces are determined as a baseline for each mouse. On Day 1, each mouse receives 0.5 ml (about 10.sup.9 CFU/ml) of VRE (e.g., ATCC 51575) suspension in Muller-Hinton broth (MHB), or MHB alone (control), via gavage with a stainless steel feeding tube. At specified intervals thereafter (e.g., 1, 7, 14 days and so on), 2 fresh fecal pellets from each mouse are collected, weighed, and emulsified in MHB and the numbers of CFU of VRE, enterococci, and gram-negative enteric bacilli per gram of feces are determined by standard serial dilution and plating techniques. For example, total enterococcal counts can be measured with bile-esculin agar, counts of enteric bacilli with MacConkey agar, and counts of VRE with Muller-Hinton II agar containing vancomycin (50 g/ml), streptomycin (100 g/ml), polymyxin (100 g/ml) and nystatin (2 g/ml) (see M. S. Whitman et al., 1996, Gastrointestinal tract colonization with vancomycin-resistant Enterococcus faecium in an animal model, Antimicrobial Agents and Chemotherapy 40(6):1526-1530). Groups of mice (at least 5 mice/group) are assigned to receive daily either sterile drinking water (control), or drinking water containing 100 g/ml, 200 g/ml or 300 g/ml of gallium citrate, 250 g/ml of vancomycin, 250 g/ml of linezolid, or 200 g/ml of gallium citrate and 250 g/ml of linezolid, starting 24 hours after the inoculation of the mice up to 10 days. Counts of VRE and total enterococci in feces are determined for each group at specified intervals up until 40 days after the inoculation and compared with the baseline counts.

Example 4

[0058] Helicobacter pylori

[0059] C57BL/6 mice are inoculated with the mouse-adapted Helicobacter pylori SS1 strain (Lee A, O'Rouke et al., 1997, A standardized mouse model of Helicobacter pylori infection: introducing Sydney strain, Gastroenterology 112:1386-97) by intragastric delivery of 0.1 ml of the bacterial suspension (approximately 1-210.sup.9 bacteria/ml) in an appropriate medium (e.g., brucella broth). Control mice are given 0.1 ml of the medium without the bacteria. Mice are left for 1-3 weeks for bacterial colonization to become established. Groups of mice (at least 5 mice/group) are assigned to receive daily, via intragastric gavage, either sterile saline (control), or 60 mg/kg, 80 mg/kg or 100 mg/kg of gallium maltolate with or without 15 mg/kg of omeprazole in saline solution for 14 days. Mice are euthanized 24 hours after the completion of the treatment. A longitudinal section of gastric tissue is removed, fixed in formalin solution, embedded in paraffin and cut at 8. to produce histologic sections. The sections are prepared with Giemsa stain and examined microscopically for Helicobacter pylori colonization of the gastric mucosa. A second longitudinal section of gastric tissue is removed, weighed and homogenized in 1 ml brucella broth. The homogenate is diluted in phosphate-buffered saline and an aliquot is plated, in duplicate, on a selective medium (e.g., blood agar supplemented with 5% defibrinated sheep blood, 100 g/ml vancomycin, 3.3 g/ml polymixin B, 200 g/ml bacitracin, 10.7 g/ml nalidixic acid and 50 g/ml amphotericin B (see J. I. Keena et al., 2004, The effect of Helicobacter pylori infection and dietary iron deficiency on host iron homeostasis: A study in mice, Helicobacter 9(6):643-650). Growth of Helicobacter pylori is confirmed based on Gram staining, morphology and urease production. The numbers of colony forming unit (CFU) per gram of tissue are determined and compared among the groups.

6. EQUIVALENTS

[0060] Those skilled in the art to which the present invention is related will recognize, or be able to ascertain, many equivalents to the specific embodiments of the invention described herein using no more than routine experimentation. Such equivalents are intended to be encompassed by the following claims.

[0061] All publications, patents and published patent applications mentioned in this specification are herein incorporated by reference into the specification.

[0062] Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.