RIFABUTIN TREATMENT METHODS, USES, AND COMPOSITIONS

Abstract

The invention provides systems and methods for increased clinical efficacy of rifabutin against A. baumannii. The invention takes advantage of the discovery of a ferric-coprogen (FhuE) receptor that is responsible for the uptake of rifabutin into A. baumannii cells. Methods preferably include obtaining a sample from a patient suspected of having an infection; performing a test on the sample to identify an infection of A. baumannii in the patient; and providing a formulation of rifabutin for treating the patient that, when administered to the patient, maximizes a resultant AUC and/or C.sub.max. The method may include administering the formulation of rifabutin to the patient. Preferably the formulation is delivered to the patient, e.g., by intravenous injection and results in a C.sub.max is that greater than about 2 mg/L and optionally less than about 50 mg/L.

Claims

1. A method of treating A. baumannii infection, the method comprising: administering to a patient a composition comprising rifabutin at a dose sufficient for activation of a ferric-coprogen (FhuE) receptor of A. baumannii cells, to thereby facilitate entry of said rifabutin into said A. baumannii cells.

2. The method of claim 1, wherein the composition is administered intravenously.

3. The method of claim 2, wherein the dose provides an AUC and C.sub.max associated with activation of the FhuE receptor.

4. The method of claim 3, wherein the C.sub.max is greater than about 2 mg/L.

5. The method of claim 3, wherein said C.sub.max is greater than about 2 mg/L and less than about 50 mg/L.

6. The method of claim 3, wherein C.sub.max>2 mg/L but <50 mg/L and AUC>10 mg*h/L and <300 mg*h/L.

7. The method of claim 1, wherein rifabutin is administered by inhalation.

8. The method of claim 1, wherein rifabutin is administered by a modified release oral drug delivery system.

9. The method of claim 1, wherein the composition comprising rifabutin includes the rifabutin, water, a solvent, and an acid.

10. The method of claim 9, wherein the composition is delivered intravenously at a rifabutin dose that is at least about 2 mg/kg q24 h, 1 mg/kg q12 h, or 0.5 mg/kg q6 h.

11. The method of claim 9, wherein the solvent is selected from the group consisting of polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monooleate polyoxyethylene sorbitan monolaurate (Tween 20), polyethylene glycol (PEG), propylene glycol, N-methyl-2-pyrrolidone (NMP), glycerin, ethanol, dimethylacetamide (DMA), diethylene glycol monoethyl ether (transcutol HP), and dimethyl isosorbide (DMI).

12. The method of claim 9, wherein the composition comprises about 2:1 solvent:rifabutin.

13. The method of claim 9, further comprising delivering the formulation to the patient by intravenous injection.

14. The method of claim 13, wherein the formulation is delivered at a dose that results in a C.sub.max is that is at least about 2 mg/L.

15. The method of claim 13, wherein the formulation is delivered at a dose that results in: 2 mg/L<C.sub.max<50 mg/L; and 10 mg*h/L<AUC<300 mg*h/L.

16. The method of claim 13, wherein the formulation is delivered at a dose that is at least about 2 mg/kg q24 h, 1 mg/kg q12 h, or 0.5 mg/kg q6 h.

17.-29. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIG. 1 is a graph illustrating the quantification of fhuE expression levels in A. baumannii HUMC1 in different media.

[0043] FIG. 2 is a graph of the activity of IV administration of rifabutin in a murine neutropenic sepsis model.

[0044] FIG. 3 shows effects of rifabutin in neutropenic lung infection mouse models with CD-1 mice infected with A. baumannii UNT091.

[0045] FIG. 4 shows effects of rifabutin in neutropenic lung infection mouse models with CD-1 mice infected with A. baumannii UNT093.

[0046] FIG. 5 provides results of dose fractionation experiments indicating a clear dose response relationship for which both C.sub.max and AUC are critical for activity.

[0047] FIG. 6 is a graph showing CFU per lung pair in mice inoculated with A. baumannii.

[0048] FIG. 7 shows a schematic of TonB-dependent transport.

[0049] FIG. 8 shows activity of rifabutin and rifampicin antibiotics upon plasmid mediated expression of FhuE-variants in CA-MHB.

DETAILED DESCRIPTION

[0050] Embodiments of the disclosure provide methods, uses, and compositions for treating, or making a medicament for treating, A. baumannii infection. For background, see Howard, 2012, Acinetobacter baumannii: An emerging opportunistic pathogen, Virulence 3(3):243-250 and Peleg, 2008, Acinetobacter baumannii: Emergence of a successful pathogen, Clin Microbiol Rev 21(3):538-582, both incorporated by reference. Methods and compositions of the disclosure operate through the activation of a ferric-coprogen (FhuE) receptor to facilitate entry of said rifabutin into said A. baumannii cells. The FhuE receptor is discussed in Sauer, 1987, Ferric-coprogen receptor FhuE of Escherichia coli: Processing and sequence common to all TonB-dependent outer membrane receptor proteins, J Bact 169(5):2044-2049, incorporated by reference.

[0051] Methods preferably include obtaining a sample from a patient suspected of having an infection; performing a test on the sample to identify an infection of A. baumannii in the patient; and providing a formulation of rifabutin for treating the patient that, when administered to the patient, maximizes a resultant AUC and C.sub.max. The method may include administering the formulation of rifabutin to the patient. Preferably the formulation includes rifabutin, water, a solvent, and an acid. Preferred solvents include polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monooleate polyoxyethylene sorbitan monolaurate (Tween 20), polyethylene glycol (PEG), propylene glycol, N-methyl-2-pyrrolidone (NMP), glycerin, ethanol, dimethylacetamide (DMA), diethylene glycol monoethyl ether (transcutol HP), dimethyl isosorbide (DMI), or another polar solvent. The formulation may about 250 mg/ml (1:1 v/v solvent/water) or about 166.7 mg/ml (2:1 solvent/water), however concentrations of the reconstituted solution may be as high as about 300 mg/ml. In certain embodiments, the formulation is delivered to the patient, e.g., by intravenous injection. Preferably, the IV injection results in a C.sub.max is that greater than about 2 mg/L and optionally less than about 50 mg/L. In some embodiments, the formulation comprises a dose of rifabutin having a C.sub.max>2 mg/L but <50 mg/L and AUC>10 mg*h/L and <300 mg*h/L. The formulation may be delivered at a dose that is at least about 2 mg/kg q24 h, 1 mg/kg q12 h, or 0.5 mg/kg q6 h. With reference to FIG. 5, the formulation is preferably delivered via IV at a dose that is at least about 2 mg/kg q24 h, 1 mg/kg q12 h, or 0.5 mg/kg q6 h.

[0052] Methods and compositions of the disclosure exploit the insight that the A. baumannii siderophore receptor FhuE plays an important role in rifabutin uptake.

[0053] FIG. 1 shows that fhuE is at least 10-fold overexpressed when A. baumannii is grown in nutrient depleted medium (Roswell Park Memorial Institute (RPMI) medium plus 10% fetal calf serum (FCS)) compared to standard testing conditions (cation-adjusted Mueller Hinton broth; CA-MHB). As shown in the Examples, deletion of fhuE resulted in elevated MICs towards rifabutin in RPMI+10% FCS however, surprisingly had no effect on the closely related compound rifampicin. These results confirmed that FhuE is required for potent rifabutin activity in RPMI+10% FCS and indicated that rifabutin activity in this medium is likely due to active uptake of the compound mediated by the A. baumannii siderophore receptor FhuE.

[0054] FIG. 2 is a graph illustrating the results of the effect of rifabutin and rifampin in a neutropenic sepsis mouse model. The results indicate that the IV administration of rifabutin protects against sepsis with a dose dependent response at 1 mg/kg whereas rifampin does not at 10 mg/kg supporting the potent activity of rifabutin observed in vitro.

[0055] FIG. 3 shows effects of rifabutin in neutropenic lung infection mouse models with CD-1 mice infected with A. baumannii UNT091.

[0056] FIG. 4 shows effects of rifabutin in neutropenic lung infection mouse models with CD-1 mice infected with A. baumannii UNT093.

[0057] FIG. 5 provides results of dose fractionation experiments indicating a clear dose response relationship for which both C.sub.max and AUC are critical for activity.

[0058] In preferred embodiments, the disclosure provides methods of treating A. baumannii infection by administering rifabutin in an amount that maximize the AUC and C.sub.max. The administration may be achieved by intravenous injection or by methods known in the art for modified oral released or by inhalation if locally high C.sub.max and AUC are required.

[0059] In certain embodiments, rifabutin is administered as an intravenous formulation in an amount that maximize the AUC and C.sub.max. By such methods, rifabutin will reach therapeutic concentrations and reduce the frequency of resistance development. For example, in preferred embodiments, C.sub.max is greater than about 2 mg/L and less than about 50 mg/L.

[0060] Embodiments of the disclosure provide a composition that includes rifabutin. In certain aspects, the disclosure provides a composition for use in treating A. baumannii infection, the composition comprising rifabutin, water, a solvent, and an acid. The solvent may be polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monooleate polyoxyethylene sorbitan monolaurate (Tween 20), polyethylene glycol (PEG), propylene glycol, N-methyl-2-pyrrolidone (NMP), glycerin, ethanol, dimethylacetamide (DMA), diethylene glycol monoethyl ether (transcutol HP), or dimethyl isosorbide (DMI). The composition may include between about 1:1 and 2:1 v/v solvent/water. Most preferably, the composition is provided within an IV bag. In preferred embodiments, the composition is provided for a patient identified as infected with the A. baumannii, and the composition includes rifabutin for IV delivery at a dosage that is at least about 2 mg/kg q24 h, 1 mg/kg q12 h, or 0.5 mg/kg q6 h. The composition may have a formulation that provides for a dose of rifabutin having a C.sub.max>2 mg/L but <50 mg/L and AUC>10 mg*h/L and <300 mg*h/L.

[0061] Preferably, the formulation is intended for intravenous delivery.

[0062] In other aspects, the disclosure provides a use of rifabutin for the manufacture of a medicament for treating A. baumannii infection in a patient, in which the medicament is prepared to be administered in a dosage regime that results in a C.sub.max in the patient that is that is at least about 2 mg/L. Preferably, the medicament comprises rifabutin, water, an acid, and a solvent (e.g., polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monooleate polyoxyethylene sorbitan monolaurate (Tween 20), polyethylene glycol (PEG), propylene glycol, N-methyl-2-pyrrolidone (NMP), glycerin, ethanol, dimethylacetamide (DMA), diethylene glycol monoethyl ether (transcutol HP), or dimethyl isosorbide). In a preferred embodiment, the solvent is DMI or trascutol HP. In certain embodiments, the solvent to water ratio v/v is 1:1 or 1:2. The acid may be hydrochloric, methanesulfonic, phosphoric, L-tartaric, D-glucuronic, L-malic, D-gluconic, L-lactic, acetic, or L-aspartic acid. In a preferred embodiment, the acid is acetic acid or D-glucuronic acid. In certain embodiments, the rifabutin to acid molar ratio is 1:1.

[0063] Most preferably, the medicament is provided within an IV bag. In preferred embodiments of the use, the dosage regime results in: 2 mg/L<C.sub.max<50 mg/L; and 10 mg*h/L<AUC<300 mg*h/L. The dosage regime may preferably result in a dose that is at least about 2 mg/kg q24 h, 1 mg/kg q12 h, or 0.5 mg/kg q6 h.

[0064] Optionally, the rifabutin may be administered by inhalation in an amount that maximize the local AUC and C.sub.max. Such methods and compositions allow the rifabutin to reach therapeutic concentrations and reduce the frequency of resistance development.

[0065] In some embodiments, rifabutin is administered by a modified release oral drug delivery system to enhance C.sub.max and AUC and minimize t.sub.max so to maximize the AUC and C.sub.max. Such systems and methods allow rifabutin to reach therapeutic concentrations and reduce the frequency of resistance development.

EXAMPLES

Example 1

[0066] A number of approved drugs were tested against A. baumannii under standard testing conditions (cation-adjusted Mueller Hinton broth; CA-MHB) as well as nutrient depleted medium (Roswell Park Memorial Institute (RPMI) medium plus 10% fetal calf serum (FCS). The antibacterial activity of rifabutin against A. baumannii was greatly enhanced under non-standard testing conditions. Table 1 summarizes the results of the antimicrobial susceptibilities of carbapenem resistant A. baumannii strains HUMC1 and UNT091 under standard (CA-MHB, Mueller Hinton broth 2) and non-standard testing conditions. The results indicate that the two carbapenem resistant A. baumannii strains, HUMC1 and UNT091, were highly susceptible towards rifabutin (MIC=0.002 mg/L) when tested in RPMI supplemented with 10% FCS, but showed low susceptibility for rifampin, meropenem, cefotaxime, gentamicin and ciprofloxacin. In striking contrast, both strains had low susceptibility towards all of the tested antibiotics (including rifabutin), when tested under standard testing conditions (CA-MHB broth).

TABLE-US-00001 TABLE 1 Antibacterial activity of rifabutin, rifampin and comparators against A. baumannii in nutrient depleted and standard testing conditions MIC (mg/L) RPMI plus 10% FCS CA-MHB cipro- cipro- rifabutin rifampin meropenem cefotaxime gentamicin floxacin rifabutin rifampin meropenem cefotaxime gentamicin floxacin HUMCI 0.002 16 >16 >64 >16 >4 4 16 >16 >64 >16 >4 UNT091 0.002 16 >16 >64 >16 >4 8 8 >16 >64 >16 >4

[0067] Methods for Testing in Example 1, Table 1.

[0068] The in vitro activity of rifabutin, rifampin, meropenem, cefotaxime, gentamicin and ciprofloxacin against two carbapenem-resistant clinical A. baumannii isolates in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% (v/v) fetal calf serum (FCS) and under standard minimum inhibitory concentration (MIC) assay conditions was analyzed.

[0069] Stock solutions of rifabutin were prepared at 2 mg/mL in DMSO and stored at −20° C.

[0070] Two A. baumannii isolates were used in this example: HUMC1 (BV374) (Spellberg/Luna Laboratory, University of Southern California, Los Angeles, Calif.) and UNT091-1 (BV378) (UNT Health Science Center, Fort Worth, Tex.). The HUMC1 isolate is a hyper-virulent drug-resistant clinical strain isolated from a blood-stream infection. Both strains are carbapenem resistant and colistin sensitive. The isolates were stored at −80° C. as 20% (v/v) glycerol cultures.

[0071] MICs were determined by the broth microdilution method following the guidelines of the Clinical Laboratory Standards Institute (CLSI) using RPMI supplemented with 10% (v/v) or cationic-adjusted Muller Hinton broth (CA-MHB) as assay medium. To prepare the bacterial inocula, 3-5 colonies of bacterial strains from over-night growth on ChromAgar orientation plates (CHROMagar Cat. No. RT412) were suspended in 5 mL saline. The turbidity of the bacterial suspension was adjusted to 0.5 McFarland units (equal to an optical density at 610 nm (OD610) to 0.08-0.1). This suspension was diluted 200-fold in RPMI supplemented with 10% (v/v) FCS to reach a final concentration of approximately 106 colony forming units (CFU)/mL and was used to inoculate the microtiter plates.

[0072] Serial 2-fold dilutions of the antibiotics were prepared in a separate 96-well plate polypropylene U-bottom plate (Ratiolab Cat. No. 6018111) in RPMI supplemented with 10% (v/v) FCS at 10-fold of the final test concentrations and 10 μl of the dilutions were transferred to new 96-well polystyrene U-bottom microtiter plates with a parafilm plate cover.

[0073] The plates were then inoculated with 90 μL per well of the prepared bacterial suspensions in using a multichannel pipette (Eppendorf), with the first column containing 4 wells each for growth control (no antibiotic). The plates were covered with parafilm and incubated at 35° C. for 20-24 h, after which the MIC was determined by visual inspection and the plates were scanned to record the data. The MIC was recorded as the lowest concentration of the compound that inhibited bacterial growth by visual inspection. MICs were determined at least in duplicates and in the case of variations, the higher values are provided.

Example 2. FhuE Overexpression: The Level of fhuE Expression was Evaluated in Different Media by qRT-PCR on the A. baumannii HUMC1 Strain

[0074] FIG. 1 depicts a graph illustrating the quantification of fhuE expression levels in different media. As depicted, fhuE is at approximately 10-fold overexpressed when A. baumannii is grown in RPMI+10% FCS or CA-MHB supplemented with 0.1 mM pyridoxal isonicotinoyl hydrazone (PIH) compared to CA-MHB. These results indicate that the increased rifabutin activity is due to the increased fhuE expression in these media.

[0075] Methods for Measuring fhuE Expression Levels, FIG. 1.

[0076] The expression of fhuE was evaluated by quantitative reverse transcription-PCR (qRT-PCR). Isolates were grown in specified broth at 37° C. to mid-log phase (optical density at 600 nm [OD600] of 0.5), and total RNA was extracted using a PureLink RNA minikit (Ambion) according to the manufacturer's recommendations. Residual DNA contaminations were removed using a Turbo DNA-free kit (Ambion). qRT-PCR was performed using a GoTaq 1-Step RT-qPCR System kit (Promega) on a CFX96 Touch™ Real-Time PCR Detection System (BioRad). As a housekeeping gene, the RNA polymerase sigma factor D (rpoD) was quantified and fhuE expression was normalized to that of rpoD using the comparative MET (where CT is threshold cycle) method.

Example 3. Deletion of fhuE

[0077] To confirm the role of FhuE activation in rifabutin activity, fhuE was deleted in the A. baumannii strains HUMC1 and UNT091.

[0078] Table 2 summarizes MICs of rifabutin in RPMI medium supplemented with 10% (v/v) FCS for the fhuE deleted mutants and their parental strains. Deletion of fhuE resulted in elevated MICs towards rifabutin in RPMI+10% FCS however, surprisingly had no effect on the closely related compound rifampicin. These results confirmed that FhuE is required for potent rifabutin activity in RPMI+10% FCS and indicated that rifabutin activity in this medium is likely due to active uptake of the compound mediated by the A. baumannii siderophore receptor FhuE. The data from this experiment show that rifabutin is very active against A. baumannii because of a novel mechanism of entry in A. baumannii.

TABLE-US-00002 TABLE 2 Antibacterial activity of rifabutin and rifampicin FhuE depleted mutants of A. baumannii. HUMC1 UNT091 Drug/Isolate HUMC1 ΔfhuE UNT091 ΔfhuE rifabutin (MIC = mg/L) 0.002 2 0.002 0.5 rifampin (MIC = mg/L) 32 32 2 4

[0079] Methods for Constructing the fhuE Deletion Mutant, Table 2.

[0080] The gene AWC45_RS10145 (HUMC1 genome) coding for the FhuE protein was deleted in the A. baumannii strains HUMC1 and UNT091 using a two-step recombination method. DNA fragments corresponding to 700-bp up and downstream genomic regions of fhuE were amplified by PCR and introduced into the pVT77 knockout plasmid using Gibson assembly. The resulting fhuE knockout plasmid was transferred in A. baumannii isolates by conjugation and trans-conjugants were selected on LB agar plates containing sodium tellurite. After overnight selection at 37° C., clones were screened for genomic plasmid integration by PCR and clones containing up- and downstream plasmid integrations were used for counter-selection on LB agar plates containing AZT for plasmid removal from the genome. Clones were screened for fhuE deletion and plasmid removal by PCR, and the genomic gene deletions were confirmed by DNA sequencing (Microsynth AG, Balgach, Switzerland).

Example 4. Plasmid-Based Expression of fhuE

[0081] Overexpression of fhuE was evaluated to determine whether it triggers rifabutin uptake.

[0082] Table 3 summarizes MICs of rifabutin in fhuE expressing A. baumannii strains in CA-MHB +/−1 mM IPTG. In the presence of IPTG, rifabutin MIC was 1000-fold lower in the strain carrying the fhuE expressing plasmid compared to the strain carrying an empty plasmid as control. This data shows that activation of fhuE in A. baumannii results in a potent activity of rifabutin towards this organism.

TABLE-US-00003 TABLE 3 MICs of rifabutin in fhuE expressing A. baumannii strains in CA-MHB +/− 1 mM IPTG rifabutin MIC (mg/L) CA-MHB CA-MHB Strain Plasmid no IPTG 1 mM IPTG A. no plasmid 4 4 baumannii empty plasmid 1 2 ATCC 17978 FhuE 0.016 0.002 expressing plasmid

[0083] Methods for overexpression of fhuE in A. baumannii, Table 3.

[0084] The fhuE gene (AWC45_RS10145) from the A. baumannii HUMC1 strain was cloned into the E. coli/A. baumannii shuttle plasmid pVT111 under the control of the isopropyl-beta-D-1-thiogalactopyranoside (IPTG) inducible promoter Ptrc-lacO. The resulting plasmid and the original pVT111 plasmid (control) were transferred to the A. baumannii strain ATCC-17978 by conjugation and transconjugants were selected on LB agar plates containing kanamycin. The presence of the plasmids in the receiver A. baumannii strains was then confirmed by PCR.

Example 5: Frequency of Mutational Resistance (FoR) Towards Rifabutin

[0085] Table 4 summaries the FoR results of the A. baumannii spontaneous resistance frequencies to rifabutin on RPMI+10% FCS agar medium. Dose-dependent FoR ranging from 10.sup.−5 to 10.sup.−9 was observed for the HUMC1 strain. High FoR's around 10.sup.−5 were observed at rifabutin concentrations of 0.02 and 0.2 mg/L, followed by a step wise decrease to 10.sup.−7 at 1 mg/L and 10.sup.−9 at 2 mg/L and 20 mg/L rifabutin. Similar results were obtained for strains UNT091-1, ACC00445, LAC-4 and UNT238-1.

TABLE-US-00004 TABLE 4 Frequency of Mutational Resistance (FoR) results of the A. baumannii spontaneous resistance frequencies to rifabutin on RPMI + 10% FCS agar medium rifabutin A. baumannii clinical isolate (mg/L) HUMC-1 UNT091 ACC00445 LAC-4 UNT238 0.02 2.20E−05 7.80E−06 1.10E−05 0.1 1.70E−05 0.2 1.70E−05 6.40E−06 8.10E−06 7.00E−05 2.30E−05 1 4.30E−07 2 3.30E−09 7.10E−09 2.40E−09 5.00E−09 8.80E−10 20 2.10E−09 7.80E−09 5.70E−09 4.20E−09 1.60E−08

[0086] The five clinical A. baumannii strains reveal a dose dependent frequency of mutational resistance reaching 10.sup.−9 at rifabutin concentrations of ≥2 mg/L. Similar in vitro FoR (10.sup.−9) have been demonstrated for other antibiotics used as standard of care to treat A. baumannii infections. The results demonstrate that rifabutin can be used to efficiently treat A. baumannii infections. Importantly, it was identified that the route of administration must achieve systemic drug concentrations of ≥2 mg/L to prevent rapid resistance development, a concentration not achievable with the currently available oral formulations.

[0087] Methods for determining the frequency of mutational resistance towards rifabutin, Table 4.

[0088] The frequency of A. baumannii mutational resistance (FoR) to rifabutin in the RPMI medium supplemented with 10% (v/v) fetal calf serum (FCS) was investigated.

[0089] Stock solutions of rifabutin were prepared at 10 mg/mL in DMSO and stored at −20° C. A. baumannii clinical isolates were stored at −80° C. as 20% (v/v) in glycerol stock cultures.

[0090] Selective agar plates were prepared using RPMI powder (Sigma R7755) dissolved at 10.3 g/L with agar at 15 g/L and boiled until complete agar melting. After cooling the media to 45° C., 0.3 g/L L-glutamine (Sigma G7513), 25 mM HEPES (Gibco 15630-056) and 10% (v/v) FCS (Gibco 10500-064) were added. Concentrations of 0.02, 0.1, 1.0, 2.0 and 20.0 mg/L of rifabutin were supplemented and 25 mL of the media was poured directly into 9 cm petri dishes.

[0091] The culture inocula were prepared from a bacterial NaCl suspension at 0.5 McFarland diluted 200-times in 100 mL of RPMI (Sigma R8758)+10% FCS in 500 mL flasks to reach ˜5×10.sup.5 CFU/mL. The flasks were incubated for 24 h at 37° C. under shaking at 220 rpm. After incubation, the cells were pelleted by centrifugation (10 min, 7000 rpm at RT) and re-suspended in 1 mL PBS. Ten-fold dilution series of the cell suspensions were prepared in PBS and 100 of the resulting cell suspension were inoculated on the rifabutin containing selection plates described above, as well as on non-selective plates to determine the cell density of the inocula. After incubation at 35° C. for 24 h colonies were counted, and the frequency of resistance was calculated as the ratio between the number of colonies growing on plates with antibiotic and the total colony count of the inocula.

Example 6: In vivo Studies

[0092] FIG. 2 is a graph illustrating the results of the effect of rifabutin and rifampin in a neutropenic sepsis mouse model (n=7). The CD-1 mice were IP infected with A. baumannii ACC00445 strain in the presence of 5 mucin. The mice were treated with rifabutin and rifampin via IV administration at 1 h and 5 h post-infection. The results indicate that the IV administration of rifabutin protects against sepsis with a dose dependent response at 1 mg/kg whereas rifampin does not at 10 mg/kg supporting the potent activity of rifabutin observed in vitro.

[0093] FIG. 3 is a graph illustrating the results of the effect of rifabutin in neutropenic lung infection mouse models (n=5/dose group) in which the CD-1 mice were intranasally infected with A. baumannii UNT091. The mice were treated with rifabutin via IV administration at 2 h post-infection.

[0094] FIG. 4 is a graph illustrating the results of the effect of rifabutin in neutropenic lung infection mouse models (n=5/dose group) in which the CD-1 mice were intranasally infected with A. baumannii UNT093. The mice were treated with rifabutin via IV administration at 2 h post-infection.

[0095] After 24 hours of treatment the mice were sacrificed and colony forming units in the lungs were measured. The results indicate the IV administration of rifabutin results in a potent effect at doses of <0.5 mg/kg supporting the potent activity of rifabutin observed in vitro.

[0096] FIG. 5 summarizes the results of dose fractionation experiments in a neutropenic mouse model of infection. Rifabutin was administered IV either once daily (q24 h), bi-daily (q12 h) or four-times within 24 hours (q6 h). The results indicate a clear dose response relationship for which both C.sub.max and AUC are critical for activity.

[0097] Table 5 is the target exposures required to treat A. baumannii infections based upon MIC. The estimations are based on efficacy models and using a sigmoid E.sub.max model with variable slope to fit the dose and PK/PD index (PDI) responses to determine the PDI values of rifabutin resulting in 1-log reduction in lung CFUs using GraphPad Prism version 5.03 (GraphPad, Inc., San Diego, Calif.). From this data it is evident that the oral administration of rifabutin will not achieve the exposures required to treat >90% of the A. baumannii isolates (MIC≤1 mg/L).

TABLE-US-00005 TABLE 5 Target exposures required to treat A. baumannii infections based upon MIC MIC (mg/L) C.sub.max (mg/L) AUC (mg*h/L) 0.125 0.32 1.44 0.25 0.65 2.87 0.5 1.30 5.74 1 2.59 11.48 2 5.19 22.96
The in vivo studies demonstrate the IV administration of rifabutin is as potent and effective in vivo as in vitro. Thus, the unexpected finding that rifabutin displays a potent activity towards A. baumannii under nutrient limiting conditions (specifically iron limiting conditions) due to the activation and uptake into the bacterial cell via the fhuE siderophore transporter allows for a potent activity in mouse models of infection. Particularly, IV formulation of rifabutin are effective against A. baumannii infections.

[0098] FIG. 6 is a graph showing CFU per lung pair in mice inoculated with A. baumannii strains. Neutropenic, female CD-1 mice (5 per group) were inoculated intranasally (t=0 hr) with equivalent titers (6.90 and 6.93 log.sub.10 CFU) of A. baumannii UNT091-1 wildtype and A. baumannii UNT091-1 ΔfhuE mutant. The treatment (single IV dose) was administered at 2-hour post-infection and bacterial burden was reported at 26 hours by determining CFU/lung. Furthermore, the effect of RBT was blunted in mice infected with the UNT091-1::ΔfhuE strain supporting the role of fhuE as mediating RBT sensitivity both in vitro and in vivo.

Example 7: The In Vitro Activity of Rifabutin was Determined Towards a Panel of Clinical A. baumannii in the Presence of an Iron Chelator Leading to Increased Siderophore Receptor Expression

[0099] Rifabutin displays a potent in vitro activity against a large panel of recently isolated and mainly XDR A. baumannii including isolates non-susceptible to colistin, and carbapenems. Complexation of free iron by PIH allows robust rifabutin susceptibility testing in nutrient rich standard MHA.

[0100] All isolates were resistant to carbapenems. Rifabutin showed excellent activity towards A. baumannii with an MIC50/MIC90 of 0.008/1 mg/L in iron-chelated nutrient rich MHA, comparable to liquid MIC determination using RPMI supplemented with FCS (MIC50/MIC90 of 0.004/2 mg/L). In contrast, in standard MHA rifabutin had only marginal activity.

[0101] Methods for Determining In Vitro Activity

[0102] A panel of 293 CRAB strains isolated from Europe (n=144), United States of America (USA) (n=99) and Asia-West Pacific (n=50) regions between 2017-2019 was used for rifabutin MIC determination. The strain panel contained isolates with 10% MDR (n=29), 86% XDR (n=253) and 4% (n=11) PDR phenotypes. Isolates were collected mainly from patients with pneumonia (59%), bloodstream infections (28%), and skin and soft tissue infections (11%) and categorized according to CLSI breakpoints as XDR when non-susceptible to ≤2 of the antimicrobial classes described by Magiorakos et al. 2011. Susceptibility testing of A. baumannii with rifabutin was performed using agar dilution method with Muller Hinton Agar (MHA) supplemented with 0.1 mM pyridoxal isonicotinoyl hydrazine (PIH), a potent nontoxic iron chelator. Comparator antibiotics were tested at CLSI standard conditions.

Example 8: Modeling FhuE TonB Effects on Rifabutin Activity

[0103] In complementation assays, FhuE V38P expression was not able to restore rifabutin potent activity compared to wildtype FhuE expression indicating that physical interaction between the FhuE TonB box and the TonB energy transducing machinery is required for rifabutin potent activity as shown in FIG. 8. These data suggest that rifabutin binding to FhuE is required to activate FhuE allosteric conformational transition enabling rifabutin active transport and potent activity against A. baumannii. FIG. 8 shows activity of rifabutin and rifampicin antibiotics upon plasmid mediated expression of FhuE-variants in CA-MHB. A. baumannii ATCC-17978 was used as host strain, gene expression from plasmids was induced with 1 mM IPTG and a plasmid that did not encode fhuE was used as control.

[0104] Methods for Measuring TonB Effects on Rifabutin Activity

[0105] Potent rifabutin activity against A. baumannii is dependent on the expression of the TonB-dependent transporter (TBDT) FhuE suggesting that rifabutin is actively translocated across A. baumannii outer membrane through FhuE. TBDT mediated active transport requires specific substrate binding to activate allosteric conformational transition of the transporter leading to the recruitment of the TonB energy transducing machinery through the so-called TonB box of the TBDT as shown in FIG. 7. See, Noinaj, N., Guillier, M., Barnard, T. J. & Buchanan, S. K. TonB-dependent transporters: regulation, structure, and function. Annu. Rev. Microbiol. 64, 43-60 (2010), incorporated herein by reference. FIG. 7 shows a schematic of TonB-dependent transport adapted from Hickman, S. J., Cooper, R. E. M., Bellucci, L., Paci, E. & Brockwell, D. J. Gating of TonB-dependent transporters by substrate-specific forced remodelling. Nat. Commun. 8, 1-12 (2017), incorporated herein by reference. TBDTs are so-called gated porins with their lumen occluded by an N-terminal plug domain preventing the substrate to transit across the outer membrane. The binding of substrate induces an allosteric rearrangement of the plug domain, releasing the Ton box into the periplasmic space, where it recruits the C-terminal domain of TonB in complex with ExbB and ExbD to form the energy transducing machinery. Linkage of the OM and IM via this non-covalent complex is required to trigger the full or partial unfolding of the plug domain, allowing the passage of the substrate. TBDT: TonB-dependent transporter, OM: outer membrane, IM: inner membrane, PG: peptidoglycan.

[0106] To investigate if rifabutin is actively transported, a FhuE V38P mutant was generated that carries a mutation in the TonB box that disrupts the interaction between FhuE and TonB. See, Cadieux, N., Bradbeer, C. & Kadner, R. J. Sequence changes in the ton box region of BtuB affect its transport activities and interaction with TonB protein. J. Bacteriol. 182, 5954-5961 (2000); Funahashi, T. et al. Identification and characterization of an outer membrane receptor gene in Acinetobacter baumannii required for utilization of desferricoprogen, rhodotorulic acid, and desferrioxamine B as xenosiderophores. Biol. Pharm. Bull. 35, 753-760 (2012); the content of each of which is incorporated herein by reference.

INCORPORATION BY REFERENCE

[0107] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

EQUIVALENTS

[0108] Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.