COMPOSITIONS AND METHODS FOR INHIBITING THE GROWTH OF

FRANCISELLA

BACKGROUND

[0001] Francisella tularensis subspecies tularensis (F. tularensis) is a remarkably infectious facultative intracellular bacterium, and the etiologic agent of tularemia, a zoonotic disease that affects a variety of mammals including humans. F. tularensis infections can be acquired via aerosol, insect bites, or inoculation onto broken skin or mucous membranes [1]. F. tularensis can be divided into four subspecies including tularensis, novicida, holarctica, and mediasiatica [2, 3]. Pneumonic infection with less than 10 colony-forming units (CFU) of F. tularensis can lead to a fatal infection, if left untreated [3], while the other three subspecies are considered less infectious or nonpathogenic to humans [3,4]. Tularemia is considered to be a re-emerging disease with recent outbreaks reported worldwide, including in the United States. Because of the ease of aerosol transmission, F. tularensis can be weaponized and could be deliberately transmitted, resulting in substantial morbidity and mortality on a large scale. It has therefore been recognized as a potential biological warfare agent and is classified as Tier 1, the highest-level bioterrorism agent classified by the U.S. Centers for Disease Control and Prevention (CDC) [4, 5].

[0002] F. tularensis can be difficult to treat because it is a facultative intracellular bacterium that targets macrophages and has several mechanisms that enable it to evade immune clearance [6]. Although a live attenuated vaccine strain (LVS) of F. tularensis has been used in humans, there is no currently licensed US vaccine. Antibiotic treatment of tularemia is limited primarily to aminoglycosides, fluoroquinolones (e.g. ciprofloxacin), and tetracyclines. Presently there is no naturally acquired resistance to these antibiotics in human or environmental isolates of F. tularensis [7]. However, constant exposure of the bacteria to increasing concentrations of ciprofloxacin in vitro can select for resistant strains, including those cross-resistant to other clinically relevant antibiotics including other fluoroquinolones and aminoglycosides [8]. Additionally, sub-species of . tularensis have different antibiotic sensitivity, making treatment more complicated [9]. Even with antibiotic treatment, many therapeutic failures and relapses have been reported [10,11]. Furthermore, antibiotic resistant strains of F. tularensis can be created for bioweapon purposes. These examples, taken together, highlight an urgent need for new drugs with novel mechanisms of action against F. tularensis. Indeed, novel therapeutic approaches have been explored in recent years [12], including use of newer antibiotics (e.g.

tigecycline, ketolides, fluoroquinolones), improving antibiotic delivery in vivo (e.g.

liposome delivery), enhancement of the innate immune response by antimicrobial peptides, host-targeted therapy [13] and combinatorial approaches with conventional antibiotics and immune adjuvants [12,14].

[0003] Previous studies showed that AR-12, a small molecule derived from the COX- 2 inhibitor Celebrex, but lacking the COX-2 inhibitory activities, displayed broad- spectrum host-directed antimicrobial activity against fungi [15], Salmonella enterica serovar Typhimurium (S. Typhimurium) and . tularensis [13,16-18] wherein bacterial burdens were significantly reduced in the host macrophage, in part, through the induction of autophagy. Several AR-12 derivatives exhibit direct antibacterial activities against methicillin-resistant Staphylococcus aureus [19], multidrug resistant tuberculosis [20], and Francisella (compound 20, herein called AR-16) [21]. AR-13, an AR-12 analog, has also been shown to have antibacterial activity, but not with respect to Francisella. See e.g.. U.S. Patent Application Publication 2013/0289004.

SUMMARY

[0004] Francisella tularensis (F. tularensis) is the causative agent of tularemia and is classified as a Tier 1 select agent. No licensed vaccine is currently available in the U.S., and treatment of naturally acquired tularemia is confined to few antibiotics. In one aspect, AR-13 exhibits direct in vitro bactericidal killing activity against Francisella, including a type A strain of F. tularensis (SchuS4) and the live vaccine strain (LVS), as well as towards the intracellular proliferation of LVS in macrophages, without causing appreciable toxicity to these host cells. In another aspect, identification of an AR-13- resistant isolate indicates that this compound has an intracellular target(s), and that efflux pumps can mediate AR-13 resistance. [0005] In the mouse model of tularemia, AR-13 treatment protected 50% of the mice from lethal LVS infection and prolonged survival time from a lethal dose of F. tularensis SchuS4. Combination of AR-13 with a sub-optimal dose of gentamicin protected 60% of F. tularensis SchuS4-infected mice from death. These data show that AR-13 is an anti- Francisella agent with bactericidal activity and can increase survival in an infected host.

[0006] In another aspect, AR-13 has direct antimicrobial activities against Francisella species with a distinct mode of action compared with AR-16. While AR-13 displays bactericidal effects, AR-16 exhibits bacteriostatic activities against LVS and . tularensis SchuS4. Without being bound by theory, examination of AR-13 resistance mechanisms in the LVS strain illustrated that decreased susceptibility to AR-13 in vitro could be mediated by efflux pumps, as an efflux pump inhibitor (e.g., carbonyl cyanide m-chlorophenyl hydrazone (CCCP), timcodar (also known as VX-853), ginsenoside 20(S)- Rh2, capsaicin, piperine, D-omithine-D-homophenylalanine-3-aminoquinoline (MC- 02,595, 2)) sensitized the AR-13 resistant mutant to AR-13.

[0007] Cytotoxicity studies revealed that AR-13 displays minimal toxicity to the human monocyte-derived macrophages (hMDMs). In vivo examination of AR-13 in a mouse model of tularemia showed that AR-13 treatment protects the LVS-infected mice from death. Combining AR-13 with a sub-optimal dose of gentamicin provided increased protection against F. tularensis SchuS4 infection.

[0008] Aspects described herein provide methods of killing Francisella in a host infected with Francisella by administering AR-13 to the host to reduce the titer of Francisella by at least about 50 percent.

[0009] In another aspect, AR-13 and an antibiotic (e.g., aminoglycosides, fluoroquinolones, and tetracyclines) can be administered to the infected host. In another aspect, the aminoglycosides can be selected from the group consisting of streptomycin, gentamicin, and amikacin. In another aspect the fluoroquinolone can be, for example, ciprofloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, levofloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, gemifloxacin, moxifioxacin, sitafloxacin, and prulifloxacin. In yet another aspect, the tetracycline is selected from the group consisting of doxycycline, minocycline, doxycycline, tetracycline, oxy tetracycline, tigecycline, chlortetracycline, lymecycline, meclocycline, methacycline, and rolitetracycline.

[00010] AR-13 is administered to the host in an amount sufficient to achieve a blood concentration of up to about 2.5 μg/ml. The antibiotic can be administered to the host in an amount sufficient to achieve a therapeutic blood or tissue concentration (e.g., the amount provided in the product label for the antibiotic, product regulatory filings or literature).

[00011] Further aspects provide methods of increasing the survival of a host infected with Francisella by administering AR-13 to the host wherein the survival of the host is increased by at least about 60% compared to an untreated host. In this aspect, an antibiotic can also be administered to the host (e.g., aminoglycosides, fluoroquinolones, and tetracyclines).

BRIEF DESCRIPTION OF THE DRAWINGS

[00012] The feature and nature of the present disclosure will become more apparent from the detailed description set forth below when taken in conjunction with the accompanying drawings.

[00013] Figure 1 provides the molecular structure of AR-12 and its derivatives AR-13 and AR-16;

[00014] Figure 2 A shows the effects of AR-13 and AR-16 on the optical densities at 600 nm (OD600) of LVS grown in 2-fold serial dilutions of AR-13 and AR-16 in mTSB as measured by a plate reader 18 h after inoculation;

[00015] Figure 2B shows the bactericidal effects of AR-13 on 1.5xl0 ⁹ colony forming units (CFU) LVS incubated in 1 ml PBS (Phosphate Buffered Saline) containing 10 μg of AR-13;

[00016] Figure 2C shows the bacteriostatic effects of AR-16 on 1.5xl0 ⁹ CFU LVS incubated in 1 ml PBS containing 10 μg of AR-16;

[00017] Figure 2D shows the bactericidal and bacteriostatic effects of AR-13 and AR- 16 on 1.5xl0 ⁹ CFU of F. tularensis SchuS4 incubated in 1 ml PBS containing 10 μg of AR-13 or AR-16F;

[00018] Figure 2E shows exemplary effects of AR-13 on the growth of F. novicida in mTSB (modified tryptone soy broth) as measured by optical density (OD);

[00019] Figure 3 shows the intracellular growth of LVS in hMDMs following AR-13 treatment;

[00020] Figure 4A shows exemplary growth curves of an LVS AR-13 resistant mutant (MT) compared to an LVS wild-type strain (WT) in the presence of various concentrations of AR-13 in mTSB;

[00021] Figure 4B illustrates the stability of AR-13 resistance in LVS MT strain by assessing viable bacteria four hours post treatment with AR-13;

[00022] Figure 5A shows that AR-13 resistant (AR-13 ^r ; MT) mutant to AR-13 is sensitized to AR-13 by adding a sub-inhibitory concentration of CCCP (4 nM);

[00023] Figure 5B shows that AR-13 resistance confers EtBr resistance in LVS. WT and MT were grown in 2-fold serial dilutions of EtBr;

[00024] Figure 5C shows that efflux pump inhibitor CCCP sensitizes the AR-13 ^r mutant to EtBr;

[00025] Figure 6A illustrates the protective effects of AR-13 in a mouse model of tularemia and provides exemplary survival curves of BALB/c mice infected by the intranasal (I.N.) route with 3xl0 ³ CFU of LVS following AR-13 treatment;

[00026] Figure 6B provides exemplary survival curves of BALB/c mice infected by the I.N. route with 10 CFU of F. tularensis SchuS4 following AR-13 treatment;

[00027] Figure 6C provides exemplary survival curves of BALB/c mice infected I.N. with 10 CFU of F. tularensis SchuS4 following AR-13 treatment;

[00028] Figures 7A-7B shows the cytotoxicity of human monocyte-derived macrophages (hMDMs) cultured in the absence or presence of AR-13 or AR-16 for 24 hours (Figure 7A) and 48 hours (Figure 7B);

[00029] Figure 8 shows that the AR-13 resistant mutant does not confer resistance to kanamycin;

[00030] Figure 9 provides an exemplary growth curve of AR-13 resistant mutant (MT) and wild-type LVS (WT) in different concentrations of efflux pump inhibitor CCCP in mTSB; and

[00031] Figure 10A shows the exemplary effects of efflux pump proteins on AR-13 resistance in F. novicida (Fn);

[00032] Figure 10B shows the exemplary effects of transposon insertions in tolC on AR-13 resistance in Fn;

[00033] Figure IOC shows the exemplary effects of tolC homolog (ftn_0779) (ftl_l 107 homolog in LVS) on increasing susceptibility of AR-13 resistant mutants to AR-13; and

[00034] Figure 10D shows the exemplary effects of a wbtH (ftl_0600 homolog in LVS) mutant on sensitivity of AR-13 resistant mutants to AR-13 (Figure 10D).

DETAILED DESCRIPTION

[00035] The disclosed methods, compositions, and devices below may be described both generally as well as specifically. It should be noted that when the description is specific to an aspect, that aspect should in no way limit the scope of the methods.

[00036] In one aspect, compounds suitable for use alone or in combination with antibiotics as described herein include, for example, AR-13 (N-{4-[5-(Phenanthren-2-yl)- 3-(trifluoromethyl)-lH-pyrazol-l-yl]phenyl}sulfuric diamide), having the following structure:

[00037] One aspect provides methods of killing Francisella in a host infected with Francisella, by administering AR-13 to the host, wherein the titer of Francisella in the host is reduced by at least about 50 percent compared to an untreated host. In another aspect, the Francisella strain is selected from the group consisting of F. tularensis SchuS4 strain (Type A) and F. tularensis LVS.

[00038] Further aspects provide methods of administering AR-13 and an antibiotic to the infected host. In one aspect, the antibiotic is selected from the group consisting of aminoglycosides, fluoroquinolones, and tetracyclines.

[00039] In yet another aspect, the antibiotic is selected from the group consisting of streptomycin, gentamicin, and amikacin, doxycycline, ciprofloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, levofloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, gemifloxacin, moxifloxacin, sitafloxacin, and prulifloxacin.

[00040] When the antibiotic is gentamicin, the host can be treated with about 0.25 mg of gentamicin/kg/day. In one aspect, AR-13 is administered to the host intranasally. Gentamicin can be administered intravenously.

[00041] In another aspect, the host is treated with from about 2.5 mg to about 5 mg AR- 13/kg/day. Alternatively, the host is treated with from about 2.5 mg to about 5 mg AR- 13/kg/day and 0.25 mg gentamicin/kg/day for at least about five consecutive days. [00042] In yet another aspect, AR-13 is administered to the host in an amount sufficient to achieve a blood concentration of up to about 2.5 μg/ml.

[00043] Further aspects provide methods of increasing the survival of a host infected with Francisella, by administering AR-13 to the host wherein the survival of the host is increased by at least about 60% compared to an untreated host. In this aspect, the Francisella strain is selected from the group consisting of F. tularensis SchuS4 strain (Type A) and . tularensis LVS.

[00044] This aspect also provid methods of administering AR-13 and an antibiotic to the infected host. In one aspect, the antibiotic is selected from the group consisting of aminoglycosides, fluoroquinolones, and tetracyclines.

[00045] The antibiotic can be selected from the group consisting of streptomycin, gentamicin, and amikacin, doxycycline, ciprofloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, levofloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, gemifloxacin, moxifloxacin, sitafloxacin, and prulifloxacin.

[00046] When the antibiotic is gentamicin, the host can be treated with about 0.25 mg of gentamicin/kg/day. In one aspect, AR-13 is administered to the host intranasally. Gentamicin can be administered intravenously.

[00047] In this aspect, the host is treated with from about 2.5 mg to about 5 mg AR- 13/kg/day. Alternatively, the host is treated with from about 2.5 mg to about 5 mg AR- 13/kg/day and 0.25 mg gentamicin/kg/day for at least about five consecutive days.

[00048] AR-13 can be administered to the host in an amount sufficient to achieve a blood concentration of up to about 2.5 μg/ml.

[00049] The aspects described herein further comprise administering an efflux pump inhibitor to the host. The efflux pump inhibitor can be selected from the group consisting of carbonyl cyanide m-chlorophenyl hydrazone, timcodar, ginsenoside 20(S)- Rh2, capsaicin, piperine, and D-ornithine-D-homophenylalanine-3-aminoquinoline.

[00050] As described herein, AR-12 derivatives were screened to identify anti- Francisella agents using a standard serial dilution method, as previously higher concentrations of AR-12 were demonstrated to have adverse effects on the mammalian cells use in the in vitro studies. Compounds AR-16 and AR-13 inhibit the growth of several Francisella subspecies with MICs of 2.5 μg/ml for F. tularensis LVS (Figure 2A), and F. tularensis SchuS4 and 5 μg/ml for F. novicida (Figure 2E) at 24-hour post- inoculation. However, as discussed below, only AR-13 had a bactericidal effect on Francisella. An inhibitory effect of AR-13 has been observed in Mycobacterium tuberculosis [20], but not in serovars of the Gram-negative bacteria Salmonella (data not shown).

[00051] AR-16 and AR-13 were used at a concentration of 10 μg/ml in PBS for bacterial killing assays. Viable bacteria were evaluated at different time points post- treatment by serial dilution, plating and enumeration. As discussed herein, AR-16 and AR- 13 have distinct modes of action: AR-13 has bactericidal activities and AR-16 has bacteriostatic effects on LVS and F. tularensis SchuS4 (Figures 2B-2D), both at log and stationary phases. AR-13 treatment (10 μg/ml) leads to an approximate 2-3.5-log decrease in CFUs of F. tularensis SchuS4 and LVS, respectively, at 8 h post-treatment (Figure 2B and D). Thus, AR-13 has significant bactericidal properties which can be used to control Francisella infection.

[00052] Since AR-13 exerts bactericidal effects on Francisella in vitro, the effects AR- 13 on the growth of LVS in its primary cellular target, human monocyte-derived macrophages (hMDMs), was examined. The cytotoxicity of AR-13 on hMDMs was tested by measuring lactate dehydrogenase (LDH) [18] release in the culture supernatants from hMDMs treated with various concentrations of AR-13 at 24 hours and 48 hours post- treatment.

[00053] AR-13 induced minimal cytotoxicity towards hMDMs at AR-13 concentration as high as 40 μΜ (Figures 7A-7B). hMDMs were then infected with LVS and treated with different concentrations of AR-13. The intracellular bacterial load was evaluated 22 hours post-treatment. As shown in the Figure 3, AR-13 (10 μΜ) has inhibitory effects on the growth of LVS in hMDMs, reducing the CFU recovered by 0.5 logs. AR-12 was used as a control as it is known to inhibit intracellular Francisella growth by the induction of autophagy [17] and reduce LVS growth approximately 1 log at 5 μΜ (Figure 3). AR-12, however, is not known to have bactericidal effects on Francisella.

[00054] Without being bound by theory, examination of the resistance of microbes to new antimicrobials may provide insight into the mechanism(s) of action of the drug as well as drug targets. Francisella s ability to develop resistance to AR-13 in vitro was examined. Stepwise selection in mTSB broth with increasing concentrations of AR-13 was performed to select for AR-13 resistant mutants of LVS.

[00055] After approximately 20 overnight passes in increasing AR-13 selective pressure, AR-13 resistant mutants were obtained. As shown in Figure 4A, a representative AR-13 resistant mutant was able to grow in the presence of 20 μΜ AR-13. Examination of the stability of an AR-13 resistant mutant was performed by passing the mutant in AR-13- free mTSB for 10 overnight passes, with several clones then chosen for bacterial killing assays.

[00056] As shown in Figure 4B, after 10 overnight passes in AR-13-free mTSB, there was no difference in viable bacteria recovered between the initial AR-13 resistant mutant (MT) and that of 10 overnight passes in AR-13-free mTSB (MT-10 passes). These data suggest that Francisella is able to develop resistance to AR-13, and the resistance is relatively stable.

[00057] The observed stable AR-13 resistance in Francisella indicated an alteration in the inherent genomic information of the resistant mutant. Comparative genomic analysis was performed between the AR-13 resistant mutant and its parent wild type strain. This analysis identified three non-synonymous mutations in the AR-13 resistant mutant. Two of the mutations were found in genes encoding for outer membrane efflux proteins

FTL l 107 and FTL 1865 (TolC) with amino acid substitution Leu236Pro and

Glu441Lys, respectively (Table 1). The other mutation was in a gene encoding for a locus (FTL 0600) that is involved in O-antigen synthesis (amino acid substitution of

Pro353Ser).

[00058] Table 1. Comparative genomic analysis of AR-13 resistant (AR-13 ¹ ) mutant and the wild-type (WT). Three non-synonymous mutations were identified in two putative efflux pumps (FTL l 107 and FTL 1865) in the genome of AR-13 ^r mutant.

[00059] Based on the above, it was hypothesized that mutations in the efflux systems would result in a "gain of function" mutation that increased efflux activity. As such, treatment with an efflux inhibitor would then sensitize the AR-13 resistant mutant to AR- 13. To test this hypothesis, experiments with AR-13 and the proton-mediated efflux pump inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP) were conducted. Bacteria were cultured in sub-inhibitory concentrations of CCCP (4nM) (Figure 5A), and various concentrations of AR-13. As shown in Figure 5 A, AR-13 resistant mutants are more susceptible to AR-13 in the presence of sub-inhibitory concentrations of CCCP (4 nM). The growth of the AR-13 resistant mutant was significantly decreased at 2.5 μΜ AR-13 and nearly completely inhibited at 5 μΜ AR-13 in the presence of CCCP while the strain grew normally at all concentrations of AR-13 (up to 10 μΜ) in the absence of a subinhibitory concentration of the inhibitor (Figure 5 A). In addition, the AR-13 resistant isolate also conferred decreased sensitivity to ethidium bromide (EtBr) mediated by TolC as previously observed [27] (Figure 5B), but does not affect sensitivity to kanamycin (Figure 8). Similar to what was observed with AR-13, a sub-inhibitory concentration of CCCP sensitized the AR-13 resistant strain to EtBr (Figure 5C).

[00060] In parallel, independent mutations in the tolC efflux genes (ftl_l 107 [ftn_0079] and ftl_1865 [ftn_1703]) and O-antigen synthesis gene (ftl_0600 [ftn_1421]) in .

novicida were examiner for susceptibility to treatment AR-13. The results show that both tolC efflux genes, but not O-antigen synthesis gene, confer intrinsic resistance to AR-13 (Figures 10A-10D). These data provide strong evidence that efflux pumps mediate AR-13 resistance in Francisella, and suggests that AR-13 has intracellular targets. [00061] AR-13 exerts bactericidal effects on Francisella (Figure 2B and D) and causes minimal cytotoxicity to hMDMs (Figure 7A). To test AR-13 for its ability to protect against F. tularensis infection in vivo, the mouse model was used. Toxicity of AR-13 in mice was first evaluated in non-infected mice treated with 10 mg AR-13/kg/day intraperitoneally (IP) for 10 consecutive days. These mice did not show any abnormal clinical signs (sickness, hair ruffling) which indicated that they can tolerate at least total 100 mg AR-13/kg administered over a 10 day period. Mice were infected by the I.N. route with a lethal dose of LVS (3x10 ³ CFUs/mouse) and then the infected mice were treated daily by I P. injection with 2.5 mg, 5 mg, or 10 mg AR-13/kg/day in 200 μΐ PEG [13] for 10 days. A PEG-only treated group was included as a control.

[00062] As shown in Figure 6A, treatment of 2.5 mg AR-13/kg/day for 10 consecutive days provided the best protective effects (50% survival) from LVS infection (total drug of 25 mg AR-13/kg). To examine if the protective effects of AR-13 from LVS infection were a result of reduced bacterial growth in vivo, mice were intranasally infected with 3xl0 ³ CFUs of LVS/mouse and treated LP. with 5 mg AR-13/kg/day from day 0. At day 4 postinfection (total drug was 20 mg/kg, which was similar to the 25 mg/kg in the survival study), mice were sacrificed and bacterial burdens in the lung were evaluated. Treatment with 5 mg AR-13/kg/day treatment resulted in an approximate 7-fold reduction in the LVS CFU count compared with PEG treated control group.

[00063] AR-13 ability to control human virulent F. tularensis SchuS4 in the mouse infection model was examined. Without treatment, mice die from day 4-6 following intranasal (I.N.) infection with 10 CFU F. tularensis SchuS4 [13]. AR-13 was also tested in combination with a sub-optimal dose of gentamicin [13]. Mice were infected with 10 CFUs of F. tularensis SchuS4/mouse via the I.N. route and then treated with 2.5 mg or 5 mg AR-13/kg/day, or with 5 mg AR-13/kg/day plus 0.25 mg gentamicin/kg/day (LP.) for five consecutive days. PEG and 0.25 mg gentamicin treated groups were included as controls. As shown in Figures 6B and 6C, AR-13 treatment prolonged survival of F. tularensis SchuS4 infected mice but did not protect mice from death. All PEG and gentamicin only treated mice died at day 5 or day 6 post-infection, respectively (Figure 6B). However, the combination of a sub-optimal dose of gentamicin with 5 mg AR- 13/kg/day protected 50% of the infected mice from death. These data indicate that AR-13 could be used as a combinational therapeutic to control Francisella infection by also administering, for example, an antibiotic (e.g., gentamicin).

[00064] In another aspect, AR-13 or the compositions described herein can be used to treat or prevent the illness and/or disease caused by infectious microbes including, but not limited to, those listed above. AR-13 can be provided to an infected patient concurrently with an antibiotic or serially in any suitable order. In another aspect, AR-13 and the antibiotic can be administered to patients simultaneously or AR-13 and the antibiotic can be formulated together. In yet another aspect, AR-13 can be formulated together with an efflux pump inhibitor as described herein. In yet another aspect, AR-13, an antibiotic, and an efflux pump inhibitor can be formulated together.

[00065] In yet another aspect, AR-13 directly kills different Francisella subspecies including F. tularensis SchuS4, LVS (Figure 2), and F. novicida (Figure 2E). In this aspect, "directly kills" means killing 99.9% of a bacterial inoculum within a 24-hour exposure period ([29]) of AR-13. In a further aspect, a time-kill kinetic effect on LVS is approximately 3 and 4 log CFU reduction at 8 hours post treatment for F. tularensis SchuS4 and LVS, respectively (See, e.g.. Figures 2B and D.

[00066] Without being bound by theory, these findings suggest that the antibacterial effect of AR-13 is likely not mediated by rapid disruption of membrane integrity, but rather by activity of AR-13 with respect to an intracellular target. In contrast, AR-16, exerts bacteriostatic mti-Francisella activity (See, e.g.. Figure 2C).

[00067] As described herein, AR-13 demonstrates in vivo mti-Francisella activity. Despite infection with a lethal dose of LVS, two of four mice treated with 2.5 mg/kg/day (total 25 mg/kg for the whole course of treatment) of AR-13 recovered and survived to the study endpoint, while none of the vehicle-treated control mice survived.

[00068] The most protective dose of AR-13 for LVS-infected mice was a total of 25 mg/kg. Treatment at this dose was used to examine protection of mice from a lethal dose of F. tularensis SchuS4, a known pathogenic strain in humans. Treatment with this dose prolonged survival of the infected mice but did not protect the mice from death (Figure 6B). However, when combined with the sub-optimal dose of gentamicin, sixty percent of the mice were protected from death while all infected mice treated with sub-optimal dose of gentamicin died (Figure 6C). F. tularensis is primarily an intracellular pathogen of macrophages, and AR-13 was shown to have limited single agent activity against

Francisella in macrophages. However, gentamicin, an antibiotic that has poor cellular penetrating activity, augmented AR-13 activity in vivo.

[00069] The term "administer" or "administered" refers to providing the compositions described herein to a patient including by the patient, a healthcare professional, a caretaker, and also includes prescribing the compositions described herein to the patient.

[00070] The compositions described herein can be administered orally, parenterally (intravenously [IV], intramuscularly [IM], depot-IM, subcutaneously [SQ], and depot-SQ), sublingually, intranasally, by inhalation, intrathecally, topically, or rectally. Dosage forms known to those of skill in the art are suitable for delivery of the compositions described herein.

[00071] AR-13 can be formulated into suitable pharmaceutical preparations such as creams and gels, for topical application; suspensions, tablets, capsules, or elixirs for oral administration or in sterile solutions or suspensions for parenteral administration, suspensions or solutions appropriate for inhalation (e.g., metered dose inhalers, dry powder inhaler, nanoparticles) and lyophilized formulations for parenteral administration. AR-13 can be formulated into pharmaceutical compositions using techniques and procedures well known in the art.

[00072] In one aspect, about 0.1 to 1000 mg, about 5 to about 200 mg, or about 10 to about 50 mg of the AR-13, or a physiologically acceptable salt or ester can be

compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, etc., in a unit dosage form as called for by accepted pharmaceutical practice. The amount of active substance in compositions or preparations comprising AR-13 is such that a suitable dosage achieving the therapeutic range indicated is obtained.

[00073] In another aspect, the compositions can be formulated in a unit dosage form, each dosage containing from about 1 to about 1000 mg, about 1 to about 500 mg, or about 10 to about 200 mg of the active ingredient. The term "unit dosage from" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.

[00074] In one aspect, one or more of AR-13 is mixed with a suitable pharmaceutically acceptable carrier to form compositions. Upon mixing or addition of the compound(s), the resulting mixture may be a cream, gel, solution, suspension, emulsion, or the like.

Liposomal suspensions may also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. In one aspect, the effective concentration is sufficient for lessening or ameliorating at least one symptom of the disease, disorder, or condition treated and may be empirically determined.

[00075] Pharmaceutical carriers or vehicles suitable for administration of AR-13 described herein include any such carriers suitable for the particular mode of

administration. In addition, the active materials can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, or have another action. The compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.

[00076] In another aspect, if AR-13 exhibits insufficient solubility, methods for solubilizing may be used. Such methods are known and include, but are not limited to, using co-solvents such as ethanol (EtOH) or dimethylsulfoxide (DMSO), using surfactants (e.g., anionic, cationic, zwitterionic, and non-ionic). Specific suitable surfactants include, but are not limited to, TWEEN, poloxamer, sodium lauryl sulfate, aluminum monostearate and dissolution in aqueous sodium bicarbonate. Derivatives of the compounds, such as salts or prodrugs, may also be used in formulating effective pharmaceutical compositions.

[00077] The concentration of the compound is effective for delivery of an amount upon administration that lessens or ameliorates at least one symptom of the disorder for which the compound is administered. Typically, the compositions are formulated for single dosage administration.

[00078] In another aspect, AR-13 as described herein may be prepared with carriers that protect them against rapid elimination from the body, such as time-release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, microencapsulated delivery systems. The active compound can be included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated. The therapeutically effective dose may be determined empirically by testing the compounds in known in vitro and in vivo model systems for the treated disorder. Such carriers include controlled release formulations, such as, but not limited to, implants and

microencapsulated delivery systems, and biodegradable, biocompatible polymers such as collagen, ethylene vinyl acetate, polyanhydrides, polygly colic acid, polyorthoesters, polylactic acid, and the like. Methods for preparation of such formulations are known to those skilled in the art.

[00079] In another aspect, AR-13 and compositions described herein can be enclosed in multiple or single dose containers. The enclosed compounds and compositions can be provided in kits, for example, including component parts that can be assembled for use. For example, AR-13 in lyophilized form and a suitable diluent may be provided as separated components for combination prior to use. A kit may include AR-13 and a second therapeutic agent for co-administration. AR-13 and second therapeutic agent may be provided as separate component parts. A kit may include a plurality of containers, each container holding one or more unit dose of AR-13 described herein. In one aspect, the containers can be adapted for the desired mode of administration, including, but not limited to suspensions, tablets, gel capsules, sustained-release capsules, and the like for oral administration; depot products, pre-filled syringes, ampoules, vials, and the like for parenteral administration; and patches, medipads, gels, suspensions, creams, and the like for topical administration.

[00080] The concentration of AR-13 in the pharmaceutical composition will depend on absorption, inactivation, and excretion rates of the active compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.

[00081] In another aspect, the active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

[00082] Oral compositions will generally include an inert diluent or an edible carrier and may be compressed into tablets or enclosed in gelatin capsules. For the purpose of oral therapeutic administration, the active compound or compounds can be incorporated with excipients and used in the form of tablets, capsules, or troches. Pharmaceutically compatible binding agents and adjuvant materials can be included as part of the composition.

[00083] The tablets, pills, capsules, troches, and the like can contain any of the following ingredients or compounds of a similar nature: a binder such as, but not limited to, gum tragacanth, acacia, corn starch, or gelatin; an excipient (e.g., any suitable filler/bulking agent) such as microcrystalline cellulose, starch, or lactose; a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate; a glidant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate, or fruit flavoring.

[00084] When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials, which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. The compounds can also be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings, and flavors.

[00085] The active materials can also be mixed or co-administered with other active materials that do not impair the desired action, or with materials that supplement the desired action. AR-13 can be used, for example, in combination with an antibiotic, antiviral, antifungal, or pain reliever.

[00086] In one aspect, solutions or suspensions used for parenteral, intradermal, subcutaneous, inhalation, or topical application can include any of the following components: a sterile diluent such as water for injection, saline solution, fixed oil, a naturally occurring vegetable oil such as sesame oil, coconut oil, peanut oil, cottonseed oil, and the like, or a synthetic fatty vehicle such as ethyl oleate, and the like, alcohols, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvent; antimicrobial agents such as benzyl alcohol and methyl parabens; antioxidants such as ascorbic acid and sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA);

buffers such as acetates, citrates, and phosphates; and agents for the adjustment of tonicity such as sodium chloride and dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass, plastic, or other suitable material. Buffers, preservatives, antioxidants, and the like can be incorporated as required.

[00087] Where administered intravenously, suitable carriers include, but are not limited to, physiological saline, phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropyleneglycol, ethanol, N-methylpyrrolidone, surfactants and mixtures thereof. Liposomal suspensions including tissue-targeted liposomes may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known in the art.

[00088] In yet another aspect, compounds employed in the methods of the disclosure may be administered enterally or parenterally. When administered orally, compounds employed in the methods of the disclosure can be administered in usual dosage forms for oral administration as is well known to those skilled in the art. These dosage forms include the usual solid unit dosage forms of tablets and capsules as well as liquid dosage forms such as solutions, suspensions, and elixirs. When the solid dosage forms are used, they can be of the sustained release type so that the compounds employed in the methods described herein need to be administered only once or twice daily.

[00089] The terms "therapeutically effective amount" and "therapeutically effective period of time" are used to denote treatments at dosages and for periods of time effective to reduce microbial burden. As noted above, such administration can be parenteral, oral, sublingual, transdermal, topical, intranasal, or intrarectal. In one aspect, when

administered systemically, the therapeutic composition can be administered at a sufficient dosage to attain a blood or tissue level of the compounds of from about 0.1 μΜ to about 20 μΜ. For localized administration, much lower concentrations than this can be effective, and much higher concentrations may be tolerated. One skilled in the art will appreciate that such therapeutic effect resulting in a lower effective concentration of AR- 13 may vary considerably depending on the tissue, organ, or the particular animal or patient to be treated. It is also understood that while a patient may be started at one dose, that dose may be varied overtime as the patient's condition changes.

EXAMPLES

[00090] Example 1

[00091] The chemical structures of celecoxib, AR-13 and AR-16 are presented in Figure 1. Gentamicin, kanamycin, ethidium bromine, and carbonyl cyanide m- chlorophenyl hydrazone (CCCP) were purchased from Sigma (St. Louis, MO). F.

tularensis SchuS4 strain (Type A), and F. tularensis LVS used in this study were described previously [13, 22, 23]. When needed, F. novicida mutant strains were obtained from BEI Resources transposon library (https://www.beiresources.org). The bacteria were cultured on chocolate II agar (CHA) plates (Becton Dickinson, Sparks, MD) or modified Tryptic Soy Broth (mTSB) or agar [24] for 48 h (F. tularensis SchuS4 and LVS) or for 24 h (F. novicida) at 37 ^° C prior to use in all experiments. Experiments involving the LVS strain were performed in a BLS2 environment.

[00092] Example 2

[00093] The susceptibilities of Francisella to AR-13, AR-16 and other antimicrobials was determined by minimum inhibitory concentrations (MICs) and bacterial killing assays. MIC assays were performed by a standard microtiter broth dilution method with an inoculum of approximately lxlO ⁶ bacteria ml as described previously [24, 25]. MICs were determined by the lowest concentration of specific antimicrobial showing complete inhibition of bacterial growth after 24 h of incubation at 37°C. For the bacterial killing assays, bacterial strains were grown at 37 ^° C in mTSB or agar by supplementing with 135 μg/ml ferric pyrophosphate and 0.1% cysteine hydrochloride at 37 ^° C for 48 hours.

Bacteria were suspended in phosphate buffered saline (PBS) to an optical density (OD) of 0.4 at 600nm, equivalent to 3x10 ⁹ CFU/ml. Approximately lxl 0 ⁹ bacteria in 1 ml PBS were incubated with 10 μg AR-13 or AR-16 or control dimethyl sulfoxide (DMSO) at 37°C. Viable bacteria at different time-points were evaluated by serial dilution and plating on mTSB agar plates or CHA plates.

[00094] Example 3

[00095] To examine the mechanism of AR-13 action, AR-13 was used as the selective agent to obtain spontaneous AR-13 -resistant (AR-13 ¹ ) mutants in vitro by stepwise selection in broth culture. 50 μΐ from an overnight culture of LVS was exposed to increasing concentrations of AR-13 in 5 ml mTSB broth with 0.25 μg AR-13/ml as a starting concentration at 37°C while shaking. 50 μΐ of this LVS grown culture was then passed into 2-fold increasing concentrations of AR-13 in 5 ml mTSB. The process was repeated until LVS was able to stably grow in 20 μg AR-13/ml (approximately 20 passes). Approximately 70 AR-13 ^r clones were selected to determine the MIC to AR-13. Two representative AR-13 ^r mutants were then passed in non-selective AR-13 free mTSB for 10 passes of an overnight culture. The stable AR-13 ^r mutants were chosen for the subsequent studies including genomic sequencing and assays regarding the mechanism of AR-13 resistance.

[00096] Example 4

[00097] Human monocyte-derived macrophages (hMDMs) were isolated from human blood via venipuncture [26] from healthy donors following a protocol approved by the Ohio State University Institutional Review Board. Written informed consent was provided by study participants [13]. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood over a Ficoll cushion (GE Healthcare Bio-Science, Piscataway, NJ). PBMCs were then cultured in sterile screw-cap Teflon wells in RPMI 1640 plus L- glutamine (Gibco-Life Technologies, Grand Island, NY) with 20% autologous human serum at 37°C in a humidified incubator containing 5% C02 for 5 days. PBMCs were then recovered from Teflon wells by chilling them on ice, re-suspending the cells in RPMI 1640 with 10% autologous serum, and allowing them to attach in 6-well or 24-well tissue culture plates for 2-3 h at 37°C in a humidified incubator containing 5% C02.

Lymphocytes were then washed away leaving the hMDM monolayers at a density of approximately 2.0x l0 ⁵ cells/well for 24-well plates for LVS infection.

[00098] Example 5

[00099] The effects of AR-13 and AR-16 on hMDM viability was assessed using a lactate dehydrogenase (LDH) assay (Roche Applied Science, Indianapolis, IN) described previously [18]. Prior to the experiment, hMDM cells were seeded into 24-well plates at 2 l0 ⁵ cells/well (3 wells per test group) with RPMI 1640 supplemented with 20% autologous serum. The medium from each well was removed and replaced with fresh 2% autologous serum in RPMI 1640 containing different concentrations of AR-13 or AR-16 dissolved in DMSO (final concentration of 0.1%) or DMSO vehicle control. The positive control received 0.1% Triton X-100 at the time of harvesting supernatants. After 24 hours or 48 hours of treatment, supernatants were collected, centrifuged to remove cells, and subjected to the LDH assay (as per manufacturer's instructions). Absorbance at 570 nm was determined via a plate reader and the cytotoxicity was calculated as a percentage compared to the positive control treated cells.

[000100] Example 6

[000101] Analysis of LVS growth in hMDMs was described previously [17] with a minor modification. LVS were grown at 37°C in mTSB or agar by supplementing with 135 ug/ml ferric pyrophosphate and 0.1% cysteine hydrochloride at 37°C for 48h. Bacteria were suspended in PBS to an OD of 0.4 at 600nm, equivalent to 3x10 ⁹ CFU/ml. hMDMs were infected with LVS at a multiplicity of infection (MOI) of 50 in the presence of 2% autologous serum in RPMI 1640 plus L-glutamine (Gibco-Life Technologies). Two hours after infection, extracellular bacteria were removed by addition of 50 μg/ml of gentamicin (Gibco-Life Technologies) to the culture medium for 1 h and then the cell monolayer was thoroughly washed three times with pre-warmed RPMI 1640 to remove unattached bacteria. Infected hMDMs were then treated with different concentrations of AR-13 in fresh culture medium containing 2% autologous serum and gentamicin at 10 μg/ml to eliminate potential re-infection by extracellular bacteria. As a control, the parental compound of AR-13, AR-12, which was previously shown to inhibit Francisella growth in macrophages via induction of autophagy, was included. AR-12 and AR-13 were dissolved at a concentration of lOmg/mL in DMSO and diluted in RPMI 1640 containing 2% autologous serum to the appropriate concentrations. At 22 hours post treatment, the infected cells were lysed with 0.1% Triton X-100 (Calbiochem, San Diego, CA) in PBS for 15 min. The cell lysates were then serially diluted with PBS and spread on CHA plates. The level of surviving intracellular bacteria was determined by enumerating CFU after 72 hours incubation at 37°C.

[000102] Example 7

[000103] Pathogen free, 6-8 week old female BALB/c mice were purchased from Harlan Sprague (IN, USA). Mice were provided food and water ad libidum, divided into groups (5 mice/group unless otherwise indicated) in sterile micro isolator cages, and allowed to acclimate for 2-3 days before challenge. All experimental procedures were carried out in strict accordance with guidelines established by The Ohio State University Institutional Animal Care and Use Committee (IACUC), and all efforts were made to minimize animal suffering.

[000104] Example 8

[000105] Intranasal (I.N.) infection of F. tularensis SchuS4 and LVS was performed as previously described [13]. Briefly, F. tularensis SchuS4 and LVS were grown on CHA plates for 48 h at 37oC. The bacteria were collected, suspended in PBS, and adjusted to an OD of 0.4 at 600nm, equivalent to 3x10 ⁹ CFU/ml. The desired concentrations of F.

tularensis SchuS4 were prepared by serial dilution. Mice were infected with 10 CFU of F. tularensis SchuS4 in 50 μΐ PBS. For LVS infection, mice were infected by the I.N. route with 3x10 ³ CFU in 50μ1 PBS. Prior to the infection, mice were anesthetized with isoflourane as approved by The Ohio State University Institutional Animal Care and Use Committee (IACUC).

[000106] Example 9

[000107] To evaluate AR-13 as a treatment for infection, survival studies were performed with F. tularensis SchuS4 and LVS strains. Mice were infected with F.

tularensis SchuS4 or LVS and treated with different doses (2.5; 5.0; or 10 mg/kg/day for LVS and 2.5; 5.0 mg/kg/day for . tularensis SchuS4) of AR-13 in 200 μΐ of polyethylene glycol (PEG) PEG400:0.9% saline: ethanol (50:35: 15) given by the intraperitoneal (I P.) route [13] from day 0 until day 10 post-infection. The infected mice were monitored for survival up to two weeks post-infection. To determine the effects of AR-13 on bacterial growth in the infected mice, 5 mice/group were infected with the LVS strain via the I.N. route and treated daily with 5 mg AR-13 in 200 μΐ PEG/kg/day from day 0. At day 4 postinfection, bacterial burdens in the lung of infected mice were determined by tissue homogenization and plating for CFU enumeration.

[000108] Example 10 [000109] Data are presented as mean ± standard deviation (SD). P-values were calculated using one-way ANOVA for multiple comparisons and adjusted with

Bonferroni's correction; *p<0.05; **p<0.01 ; ***p<0.001 ; NS, not significant. Statistical analysis was performed using GraphPad Prism 6. A Chi-square test was used for survival analysis. Statistical analysis was performed using GraphPad Prism 5.

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[000145] Not every element described herein is required. Indeed, a person of skill in the art will find numerous additional uses of and variations to the methods described herein, which the inventors intend to be limited only by the claims. All references cited herein are incorporated by reference in their entirety.