WO2012050826A1

WO2012050826A1 - Methods for treating clostridium difficile infections

Info

Publication number: WO2012050826A1
Application number: PCT/US2011/053441
Authority: WO
Inventors: Richard Lee; Julian G. Hurdle
Original assignee: St. Jude Children's Research Hostpital
Priority date: 2010-09-29
Filing date: 2011-09-27
Publication date: 2012-04-19

Abstract

The present invention includes methods for treating a C. difficile infection in a patient. The methods of the present invention are based on administration of reutericyclin or reutericyclin analogs in order to kill C. difficile organisms and thus alleviate the signs and symptoms of C. difficile infection.

Description

METHODS FOR TREATING CLOSTRIDIUM DIFFICILE INFECTIONS Introduction

[0001] The patent application claims the benefit of priority from U.S. Provisional Application Serial Number 61/387,623 filed September 29, 2010, the content of which is incorporated herein by reference in its entirety.

[0002] This invention was made with government support under Grant Nos . R01AI079653 and R01AI062415 awarded by the National Institutes of Health. The government has certain rights in the invention.

Background of the Invention

[0003] Hospital-acquired Clostridium diff cile-associated diarrhea (CDAD) is becoming a major public health problem worldwide (Hookman, P. and J.S. Barkin. 2009. World J. Gastroenterol. 15:1554-1580; Freeman, J. et al . 2010. Clin. Microbiol. Rev. 23:529-549) . In the United States, there are an estimated 500,000 reported cases each year resulting in approximately 3.2 billion USD in health care costs (O'Brien, J. A. et al . 2007. Infect. Control Hosp. Epidemiol. 28:1219-1227) . C. difficile is a gram positive, anaerobic organism found naturally in the human colon. In recent times the emergence of hypervirulent strains of the C. difficile belonging to the toxinotype III (Bl/NAPl/027) group has led to an increase in the disease severity of CDAD and the occurrence of cases in community settings among healthy patients without hospitalization and antibiotic use (Freeman, J. et al . 2010. Clin. Microbiol. Rev. 23:529-549) . The enhanced virulence of Bl/NAPl/027 strains is reportedly due to increased production of toxins A and B, which are responsible for intestinal inflammation ( arny, M. et al . 2005. Lancet 366:1079-1084) . These toxins are synthesized by cells in their mid-log and stationary phase of growth. There are few treatment options for CDAD, with metronidazole or oral vancomycin being the primary choices (Aslam, S. et al . 2005. Lancet Infect. Dis. 5:549- 557) . Previously, these drugs were associated with 95% efficacy rates, but they now appear to be less effective in treating contemporary cases of severe CDAD (Pepin, J. et al. 2007. Am. J. Gastroenterol. 102:2781-2788; Pepin, J. et al. 2005. Clin. Infect. Dis. 40:1591-1597). Furthermore, vancomycin and metronidazole are not effective against clostridial spores and stationary phase bacteria. As a result, new therapeutic strategies are needed. Importantly, if new drugs active against CDAD are able to affect growth of stationary phase cells, then the effects of toxins produced by C. difficile could be mitigated. Such discoveries would indeed be valuable in view of the increasing number of cases of C. difficile in the United States and worldwide.

[ 0004 ] Live probiotics are being considered as alternative therapies for persistent CDAD (Parkes, G.C. et al . 2009. Lancet Infect. Dis. 9:237-244). However, there are disadvantages to using probiotics for treating CDAD, such as the possibility of bacteraemia or fungaemia (Rupnik, M. et al. 2009. Nat. Rev. Microbiol. 7:526-536) and the fact that in certain patients, especially immunocompromised individuals, antibiotic use is required which could potentially affect the cellular viability of the coadministered probiotics. Few studies characterize antimicrobial metabolites that are derived from probiotic organisms or determine whether these metabolites could be used to effectively kill C. difficile (Rea, M.C. et al . 2010. Proc. Natl. Acad. Sci. USA 107:9352-9357; Rea, M.C. et al. 2007. J^". Med. Microbiol. 56:940-946) . The development of such novel agents would avoid the problem of killing probiotic organisms, while ensuring that clearance of C. difficile is achieved. The metabolite molecules could also be optimized chemically, to improve their therapeutic properties and be administered in various oral forms, such as nutritional supplements and medicaments.

[ 0005 ] The organism Lactobacillus reuteri is a naturally occurring organism in human gut flora. It is also found in sourdough foods and is widely used in probiotic supplements (Kamiya, T. et al . 2006. Gut 55:191-196) . Various reports indicate that strains of L. reuteri, isolated from sourdough, produce metabolites with antimicrobial properties. One such metabolite is the lipophilic tetramic acid reutericyclin that has been shown to exhibit activity against Gram-positive pathogens (Yendapally, R. et al . 2008. J. Med. Chem. 51:1487-1491; Ganzle, M.G. et al . 2000. Appl . Environ. Microbiol. 66:4325-4333), by depolarizing the bacterial membrane of target organisms (Ganzle, M.G. 2004. Appl. Microbiol. Biotechnol . 64:326-332) . However, reutericyclin was found to be chemically unstable for therapeutic development, leading to modification of the chemical scaffold yielding stabilized mimics (Yendapally, R. et al. 2008. J^". Med. Chem. 51:1487-1491; Hurdle, J.G. et al. 2009. Antimicrob. Agents Chemother. 53:4028-4031) . The structure of stabilized reutericyclin mimic compounds is disclosed in U.S. Patent Application No. 2009/0069406, as well as the use of these compounds to kill gram negative bacteria that included Mycobacterium tuberculosis, Escherichia coli, Staphylococcus aureus, Eneterococcus faecalis, Bacillus anthracis, Bacillus subtilis, Pseudo onas aeruginosa, Streptococcus pyrogenes,

Propionbacterium acnes, and Streptococcus pneumoniae. Yet, there are no reports regarding the activity of these compounds against gram positive bacteria, including C. difficile .

Summary of the Invention

[0006] A method for killing C. difficile organisms comprising contacting C. difficile organisms with reutericyclin or a reutericyclin analog compound, and detecting the killing of C. difficile organisms. In one embodiment the C. difficile organisms are stationary phase organisms .

[0007] Another object of the present invention is a method for treating C. difficile-associated diarrhea which comprises administering to a patient with C. difficile- associated diarrhea an effective amount of reutericyclin or a reutericyclin analog compound in a pharmaceutically acceptable vehicle. In one embodiment the patient is administered an effective amount of reutericyclin or a reutericyclin analog in combination with an anti-infective agent .

[0008] Yet another object of the present invention is a method for preventing relapse of C. difficile infection which comprises administering to a patient with Clostridium difficile infection an effective amount of reutericyclin or a reutericyclin analog compound in a pharmaceutically acceptable vehicle so that relapse of C. difficile infection is prevented. In one embodiment the patient is administered an effective amount of reutericyclin or a reutericyclin analog in combination with an anti-infective agent . Brief Description of the Drawings

[0009] Figure 1 depicts the concentration-dependent killing of stationary phase BA1803 NAPl by Analog 867 at 0.25, 1, 4 and 16 ^g/ml as compared to vancomcyin (Van) and metronidazole ( tz) , each at 1 and 64 μg/ml .

Detailed Description of the Invention

[00010] It has now been found that reutericyclin and lipophilic chemically stabilized analogs of reutericyclin are bactericidal against exponential and toxin-producing stationary phase cultures of C. difficile . These findings hold important implications for the management of CDAD, a disease that is growing in severity and frequency. The discovery of the activity of this novel class of antibiotics against C. difficile is also important in that these drugs have been found to have activity to kill even persistent forms of the C. difficile organism. Moreover, experiments show that the lipophilic, chemically stabilized analogs of reutericyclin exhibit a lack of toxicity against gut epithelial cells, a particularly desirable property for new antibiotic drugs targeting gastrointestinal infections. Accordingly, the present invention provides methods for killing C. difficile and treating a CDAD by administering a lipophilic chemically stabilized analogue of reutericyclin orally as either a nutritional supplement or as an oral formulation. The ability of the analogs of reutericyclin to kill stationary phase, toxin-producing C. difficile cells can counteract toxin production that is responsible for the symptoms and disease severity of CDAD. Thus, the method of the present invention is superior to methods that employ- existing antibiotics for treatment of CDAD.

[00011] For the purposes of the present invention, a "reutericyclin analog" is a compound that shares the tetramic core of reutericyclin, is lipophilic, and is chemically stabilized. In particular embodiments, a reutericyclin analog possesses a smaller more synthetically tractable acyl substitution at the 3 -position of the tetramic core of reutericyclin and larger N-alkyl and N- aryl substitutions to the tetramic core. Exemplary reutericyclin analogs are described herein and in U.S. Patent Application No. 2009/0069406, which is incorporated herein by reference and which teaches the synthesis of lipophilic chemically stabilized analogues of reutericyclin. Reutericyclin analogs can be synthesized according to conventional methods (Yendapally, R. et al . 2008. J. Med. Chem. 51:1487-1491) and activity of said analogs can be determined as described herein. The structures of the compounds described herein are shown below .

Reutericyclin Analog 867

Analog 1138 Analog

[00012] Experiments were first performed to examine the activity of reutericyclin, analog 867 and analog 1138 against stationary phase C. difficile cells, since organisms in the stationary phase are the ones responsible for toxin production and the symptoms of CDAD . It had previously been shown that various analogs of reutericyclin were able to eradicate mature staphylococcal biofilms that were refractory to killing by most antibiotics (Hurdle, J.G. et al . 2009. Antimicroh. Agents Chemother. 53:4028- 4031) . Because there is a real need for novel therapeutics to treat infections of C. difficile, in particular CDAD, the activity of reutericyclins was examined first in vitro in well-established screening methods for identifying compounds with bactericidal or antibiotic activity. The most common quantitative measures of the in vitro activity of antibiotics are the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) . The MIC is the lowest concentration of a drug that results in inhibition of visible bacterial growth (i.e., inhibition of the growth of bacterial colonies on a plate) . The MBC is the lowest concentration of a drug that kills 99.9% of the original bacterial inoculum in a given time. The MICs and MBCs were thus determined for reutericyclin, analog 868 and analog 1138, as well as two positive control agents, vancomycin and metronidazole. Both vancomycin and metronidazole are known to have bactericidal activity against C. difficile . The C. difficile strains used in vitro included strains 9689 (toxinotype 0) , BA-1803 (toxinotype III, Bl/NAPl) , BA-1875 (toxinotype V, B1/NAP7) are representative of common strains in North America. BAA- 1803 and BAA- 1875 carry deletions in the regulator gene tcdC and therefore constitutively produces toxins A and B (Warny, M. et al . 2005. Lancet 366:1079-1084; Spigaglia, P. and P. Mastrantonio . 2002. J. Clin. Microbiol. 40:3470- 3475) . All strains were grown in pre-reduced TY broth or Brucella agar plates, at 37°C in an Anoxomat Mart II system (Mart Microbiology) .

[ 00013 ] As shown in Table 1, the MIC activities of reutericyclin and its analogues (867 and 1138) were comparable to the first-line antibiotics metronidazole and vancomcyin. In the context of the present invention a "first-line" antibiotic is one that is the drug of choice currently for treatment of the infection.

high density culture = 10⁸ to 10⁹ CFU/ml

[ 00014 ] As shown in Table 1, the reutericyclins killed log- phase cells (>3 log reduction in cells) at concentrations close to their respective MICs (i.e. ≤ 4-fold), which indicates these agents are primarily bactericidal . This finding was not consistent with the activity of these same compounds against S. aureus growing cells that required concentrations up to 32 -fold higher than the MIC to reduce the cell population to 2.7-2.9 logs in 24 hours (Hurdle, J.G. et al . 2009. Antimicrob. Agents Chemother. 53:4028- 4031) . The control agents, vancomycin and metronidazole, were also bactericidal against C. difficile .

[ 00015 ] The potent antibacterial action of reutericyclins was retained against stationary phase cultures, with killing occurring at concentrations 2-8 fold above the respective MICs for each compound tested (reutericyclin, analog 867, analog 1138) and the demonstrated activity of these compounds was far superior to both vancomycin and metronidazole (Table 1) . Importantly, the bactericidal action of compounds was independent of genetic background, since all three test strains from different toxinotypes, were equally susceptible to killing by reutericyclins as shown by their MBCs. In all cases, there was no change in the optical densities of stationary phase cultures between time 0 and 24 h at the MBC, which suggest that killing does not involve cell lysis. This finding is in agreement with previous studies which have shown that these compounds did not lyse the membranes of staphylococcal cells (Hurdle, J.G. et al . 2009. Antimicrob . Agents Chemother. 53:4028- 4031) . The unusual finding for the killing of stationary phase C. difficile is a unique property not currently exhibited by many established classes of antibiotics against several pathogenic organisms. Indeed, as shown in Table 1, non-growing stationary phase cultures of all test strains of C. difficile were inherently resistant to killing by either vancomycin or metronidazole. Vancomcyin was completely inactive at high concentrations (64 μg/ml) , while metronidazole only exhibited some killing at concentrations that were significantly higher than those required to kill log phase cultures (Table 1, Figure 1) .

[00016] Time kill studies were also performed. These studies showed that the three tested reutericyclins rapidly killed stationary phase cells of C. difficile, in a manner dependent on the concentration and not associated with cell lysis. Within 2 hours, Analog 867 at a concentration of 16 g/mL reduced the bacterial cell densities of BA-1803 cultures by an average of 3 log units (Figure 1) , reaching the limit of detection (10² CFU/ml) in 6 hours. At a 4-fold lower concentration of 4 μg/ml, a 2.9 log reduction occurred in 2 hours, but required more than 6 hours of exposure before the limit of detection was reached. Killing in excess of 3 logs was reached in 4 hours at a low concentration of 1 ^g/ml, while at 0.25 μg/ml a maximum kill of 2.3 logs was achieved in 24 hours. Similar, findings were observed against C. difficile strain 9869. These results showed that reutericyclins exhibited rapid concentration-dependent killing activity. Furthermore, throughout the 24 hour test period, vancomcyin was completely inactive (Figure 1) and metronidazole only caused a 3.5 log reduction in cells at a concentration of 64 μg/ml . The potent activities against stationary phase cells however was not maintained as reutericyclins and comparator agents both lacked sporicidal activity at the highest concentration tested (64 μg/mL) ; in contrast a 10% bleach solution was sporicidal and killed 10³ spore units within 20 minutes of exposure.

[00017] Since the reutericyclins are highly lipophilic, it is believed that they would be non-absorbable , which would engender high local concentrations in the gastrointestinal tract, permitting rapid killing of C. difficile cells. To evaluate these effects, drug absorption in the human Caco2 cell line was used as a model system. Studies using this model have been shown to closely mimic findings that occur within the human gut (Cheng Li, S.W. et al . 2008. In: Drug Absorption studies: In Situ, In Vitro and In Silico Models. Ehrhardt C. et al . (eds.) Springer: New York, pp. 418-429) . As shown in Table 2, chemical derivatives exhibited various permeability properties dependent on the N-substituent .

TABLE 2

a A ratio of greater than 2 indicates the compounds are substrates for efflux.

[ 00018 ] The analogs 867, 1135 and 1141 (a stereoisomer of 867) were poor at crossing the apical to basal compartment as shown by their low apparent permeability (Papp A/B) . The low permeability for these agents appears to result from being substrates of P-glycoprotein transporters (P-gp) that are present on Caco2. As a result, efflux of analogs 867, 1135 and 1141 contributes to their inability to be absorbed. Thus, molecules that poorly absorb are preferred as they can likely reach high local concentrations in the intestine permitting localized killing of C. difficile . In contrast, both reutericyclin and 1135 were not substrates of P-gp and could be absorbed into Caco2 cells, in a manner similar to vancomycin and metronidazole. This does not mean that these agents would have less activity in vivo per se, but rather exemplifies the potential to chemically modulate the N-substituent to obtain a range of molecules with different permeabilities for treating C. difficile infections .

[ 00019 ] Further support was found in the results from permeability assays that were performed in artificial lipid membranes. Reutericyclin and its derivatives were all able to permeate the lipid membrane as a result of their lipophilic properties (Table 3) . These data further supported the mechanism where active efflux is responsible for differences in antibiotic drug absorption, since artificial membranes lack biological transport systems and only model the physiochemical properties of drug absorption across cell membranes.

TABLE 3

Permeability of Various Test Compounds in Artificial Lipid Membranes (pH = 7.4)

Test Agent Permeability Mean %R + SD

Mean + SD

Analog 1135 261.5 + 29.7 95 + 1

Analog 1138 89.8 + 13.0 75 + 2

Analog 1141 96.9 + 56.0 87 + 2

Reutericyclin 183.6 + 11.9 93 + 0

Analog 867 560.1 + 405.5 97 + 1

Metronidazole 0 8 + 1

Vancomycin 6.2 + 6.2 8 + 7 [00020] None of the analogs tested were found to be cytotoxic against this cell line, since the IC₅₀ for each drug was higher than the maximum concentration tested (i.e. at 200 g/ml, more than 80% of cells were present) . By adding the test serum (10%) to TY broth, there was a reduction in MIC, with all compounds (Table 1) having a MIC of 2 g/mL. This concentration is still bactericidal and low enough to be reached upon therapeutic administration. The shift in MIC resulted from reutericyclins being serum bound .

[00021] Thus, the results described for the reutericyclin analogs in vitro provide evidence that reutericyclin analogs can kill C. difficile organisms in vivo. Accordingly, the present invention is a method of killing or reducing the growth of C. difficile organisms by contacting a C. difficile organism with reutericyclin or a reutericyclin analog compound. In one embodiment, the method of the present invention is a method of killing stationary phase C. difficile organisms. Further, the present invention is a method for treating CDAD in a patient which involves administering to a patient with CDAD an effective amount of reutericyclin or a reutericyclin analog compound in a pharmaceutically acceptable vehicle. In the context of the present invention, "an effective amount" of reutericyclin or a reutericyclin analog compound is that amount of the drug that has been shown to have statistically and/or biologically significant activity either in vitro or in vivo.

[00022] One of skill in the art will also appreciate that following treatment for CDAD, patients may often relapse. Although the microbiological basis of relapse is not well understood, it is thought that it may involve the survival and outgrowth of spores, biofilms or unkilled stationary phase cells. Spores are formed in late stationary phase and are susceptible to killing after they germination. Since reutericyclins have been show in the present invention to have the ability to kill stationary phase cells, these compounds may hinder the formation of spores and consequently lower potential relapse of infection via both spores and unkilled stationary phase cells. As agents that disrupt the function of the membrane, reutericyclin and its analogues would inhibit macromolecular synthesis, including the synthesis of proteins. Protein synthesis is required for spore formation, since its inhibition decreases sporulation (Ochsner et al . 2009. J Antimicrob Chemother 63:964) and further supports anti-sporulation properties for reutericyclin and analogues. Reutericyclin and its analogues also kill staphylococcal biofilms. If biofilms are responsible for C. difficile infection relapse (Rupnik et al . 2009. Nat Rev Microbiol 7:526), the action of reutericyclins against this cell type would also prevent relapse of infection. Therefore also contemplated by the present invention is use of reutericyclin and its analogs to prevent relapse of CDAD by inhibiting sporulation, spore germination and killing biofilms.

[ 00023 ] One of skill in the art would also appreciate that reutericyclins and its analogs can be used in combination therapy to treat C. difficile infection. Combinations with anti-infective agents proposed for use with reutericyclin and its analogs would be chosen by one of skill in the art based on individual patient considerations. Such anti- infectives used in combination with reutericyclin and its analogs would include but not be limited to any available antibiotic, glycopeptides (Vancomycin, Oritavancin, Telavancin) , lipoglycopeptides (e.g. Daptomycin or related antibiotics), nitroaromatic antibiotics (e.g. metronidazole, nitazoxanide or antibiotics classified as nitrofurans or nitroimidazoles), macrolides (e.g. Fidaxomicin) , Fusidic acid or Rifamycins (e.g. Rifaximin, Rifalazil) or lantibiotics (e.g. Actagardine, Actagardine) . Reutericyclins and its analogs may also be used in combination with biotherapeutics such as probiotics or probiotics, and with toxin binding polymers (e.g. tolevamer) . Combination treatments may also be necessary where reutericyclin or its analogs are used as a prophylaxis to suppress C. difficile infection in combination with a broad spectrum antibiotic being used to treat a systematic infection.

[00024] Effectiveness of the reutericyclin or a reutericyclin analog compound in killing of C. difficile organisms can be detected both in vitro and in vivo. For example, as already described above, MIC levels for C. difficile organisms can be determined in the presence and absence of reutericyclin compounds. When the method is an in vivo method of killing C. difficile in an animal of human, the method of detection can involve simply monitoring for the lack of diarrhea or a reduction in the level of diarrhea in the animal or human, wherein killing of C. difficile is known to be associated with a decrease in diarrhea in vivo (Aslam, S. et al . 2005. Lancet Infect. Dis. 5:549-557) .

[00025] The efficacy of the reutericyclin compounds has been shown herein based on the use of the in vitro screening methods. However, one of skill would understand that the results of the in vitro antibiotic activity assays described herein are directly applicable to demonstrating in vivo efficacy of antibiotics. There are many different in vivo model systems that can be used by one of skill in the art to further demonstrate efficacy and aid in identification of doses that will be both safe and effective in humans. Such animal model systems are well- accepted and used during development of new human pharmaceuticals that will undergo scrutiny by various regulatory bodies worldwide and approved for use in humans . Examples of such model systems include but are not limited to models described in the published literature. For example, a mouse model of CDAD has been provided by Chen et al. (2008. Gastroenterology 135:1984-1992). They describe a model that is claimed to closely represent human disease and as such would be useful for testing the in vivo efficacy of the reutericyclin compounds. Another model is provided in mice as described by Kaur et al . (2010. J^". Gasteroenterol . Hepatol. 25:832-838) . In such models, drugs can be tested against infections where the infection established is from inoculation of the animal with C. difficile . Demonstration of efficacy in such models is measured in many ways and would include but not be limited to a reduction in signs such as diarrhea, a reduction in bacterial cell counts determined by microscopic examination of tissue or blood samples taken from the animals, or a reduction in histopathological signs of inflammation.

[ 00026 ] It is contemplated that one of skill in the art will choose the most appropriate in vivo model system. The medical literature provides detailed disclosure on the advantages and uses of a wide variety of such models.

[ 00027 ] Once a test drug has shown to be effective in vivo in animals, clinical studies can be designed based on the doses shown to be safe and effective in animals. One of skill in the art will design such clinical studies using standard protocols as described in textbooks such as Spilker (2000. Guide to Clinical Trials. Lippincott Williams & Wilkins: Philadelphia) .

[00028] The methods of the present invention contemplate oral administration of the reutericyclin compounds. As such, one of skill would choose the formulation based on the wide variety of pharmaceutically acceptable formulations. Pharmaceutical or nutritional supplement preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatine, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose , sodium carboxymethyl - cellulose, and/or polyvinylpyrrolidone (PVP) . If desired, disintegrating agents may be added, such as the cross- linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more therapeutic agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee -making , levigating, emulsifying, encapsulating, entrapping or lyophilizing processes .

[00029] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses .

[00030] Pharmaceuticals or nutritional supplements which can be used orally include push-fit capsules made of gelatine, as well as soft, sealed capsules made of gelatine and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added .

[00031] The following non-limiting examples are provided to further illustrate the present invention.

EXAMPLES

EXAMPLE 1: Analog Synthesis, Bacterial Strains and Culture Conditions

[00032] Analogues of reutericyclins were synthesized as previously described (Yendapally, R. et al . 2008. J. Med. Chem. 51:1487-1491) . Control antibiotics metronidazole and vancomycin were purchased from Sigma-Aldrich. All organisms were obtained from American Type Culture Collection (ATCC; Manassas, VA) . The strains 9689 (toxinotype 0) , BA-1803 (toxinotype III, Bl/NAPl) , and BA-1875 (toxinotype V, B1/NAP7) are representative of common strains in North America. All strains were routinely grown in pre-reduced TY broth or Brucella agar plates, at 37°C in an Anoxomat Mart II system (Mart Microbiology) .

EXAMPLE 2 : Determination of Minimum Bactericidal

Concentrations (MBCs)

[00033] MBCs against log phase (low density) cultures were determined against a bacterial inocula of 10⁶ CFU/ml. Briefly, bacteria inocula were prepared in pre-reduced TY broth and added to 24 -well plates (Costar, Corning Incorporated) yielding final antibiotic concentrations of 16 to 0.0125 μg/ml . Following incubation for 24 hours, the lowest concentration of antibiotic that prevented visible bacterial growth was recorded as the "minimum inhibitory concentration" or MIC. Subsequently, bacteria were enumerated by plating serial dilutions of culture onto Brucella agar. Plates were incubated for up to 48 hours before the MBC against log cells was recorded as the lowest concentration of antibiotic that killed 99.9% (≥ 3 log reduction) of cells present in the starting inoculate (determined by initial viable counts) . MBCs against day old stationary phase cells were evaluated using a bacterial inoculate of 10⁸-10⁹ CFU/ml in 24 well Costar plates, with antibiotic concentrations ranging from 64-0.031 g/ml. After incubation for 24 hours, viable counts were performed on Brucella agar and MBCs scored as described above. All MBCs were determined at least twice. Plating was done on Brucella agar to avoid the effect of carry over antibiotic, since serum contents reduce the activity of reutericyclins (Hurdle, J.G. et al . 2009. Anti icrob. Agents Chemother. 53:4028-4031) . This effect was confirmed by spiking a plate with test concentration of drug present in dilutions and plating 100 μΐ of a culture containing 10³ CFU/ml and comparing this to the unspiked culture; no statistical difference in CFU were observed. Similar results were obtained for vancomycin and metroidazole .

EXAMPLE 3 : Time Kill Studies

[00034] After exposing stationary phase cultures to various concentrations of antibiotics or drug free controls, viable counts and OD₆oonm readings were performed on samples in 24 well plates at 2 , 4, 6 and 24 hours. Serial dilutions (10^"1, 10^~3, 10^"5 and lO^-6) were plated. The detection limit was determined to be 10 CFU/plate or 100 CFU/mL . All time kill experiments were performed on two independent replicates.

EXAMPLE 4: Sporicidal Activity

[00035] The activity of antibiotics against C. difficile spores (10³ spores/ml) were determined in physiological saline (0.9% NaCl) containing antibiotic from 64-0.031 g/ml and exposure at 37°C for 24 hours. Bacteria (200 ml) were cultured in a sporulation media of Brain Heart Infusion broth containing Yeast Extract (1%) and L-cysteine (0.1%) for 10 days. Subsequently, cells were collected by centrifugation (3, 700 g, 10 min, 4°C) , washed and then resuspended to a volume of 1 ml in phosphate-buffered saline. Cells were heat inactivated at 80°C for 10 minutes, centrifuged (21, 000 g for 5 minutes) and resuspended in 70% ethanol to further remove vegetative cells. Spores were confirmed microscopically by staining with 5% malachite green/safranin solution and viable counts were determined on the stock by plating on Brucella agar supplemented with 5 mg/L of Lysozyme .

EXAMPLE 5: Intestinal Permeability in Caco2 Cells

[00036] High throughput Caco-2 permeability was performed in a 96 -well Transwell system with a modified method (Uchida, M. et al. 2009. J. Pharmacol. Toxicol. Methods 59:39-43) . Caco-2 cells were maintained at 37°C in a humidified incubator with an atmosphere of 5% C0₂. The cells were cultured in MEM containing 20% FBS in 75 cm² flasks, 100 units/ml of penicillin, and 100 g/ml of streptomycin. The Caco-2 cells were seeded onto inserts of a 96-well plate at a density of 0.165xl0⁵ cells/insert and cultured in the MEM containing 10% FBS for 21 days. Each cultured monolayer on the 96-well plate was washed twice with HBSS/HEPES (10 mM, pH 7.4) . The permeability assay was initiated by the addition of each compound solution (50 ymol/L) into inserts (apical side, A) or receivers (basolateral side, B) . The Caco-2 cell monolayers were incubated for 2 h at 37 °C. Fractions were collected from receivers (if apical to basal permeability) or inserts (if basal to apical permeability) , and concentrations were assessed by UPLC/MS (Waters; Milford, MA) . The A→B (or B→A) apparent permeability coefficients (Pappa, cm/s) of each compound were calculated using the equation, Pappa=dQ/dt l/AC₀. The flux of a drug across the monolayer is dQ/dt ( mol/s) . The initial drug concentration on the apical side is C₀ (μπιοΙ/L) . The surface area of the monolayer is A (cm²) . EXAMPLE 6: Parallel Artificial Membrane Permeability

Assay (PAMPA)

[00037] This assay is designed to analyze permeability of various compounds on a homogeneous artificial lipid membrane using the normal Double-Sink PAMPA protocol. To start, 6 ml of 10 mM compounds solution in DMSO was applied to each well in a stock plate. Compounds were diluted 200 fold in system solution buffer (SSB, pH=7.4; pION INC, Woburn, MA) . Then, 180 ml of diluted solution was added to a donor plate (pION INC, Woburn, MA) . A filter plate (acceptor plate; pION INC, Woburn, MA) containing 200 ml of acceptor sink buffer (ASB, pH=7.4; pION INC, Woburn, MA) was then placed over the donor plate. The plates were incubated at room temperature for 0.5 hour with magnetic stirring in each individual well to allow the compounds to cross the membrane. Fractions were collected from both the donor plate and the acceptor plate, and concentrations were assessed by UV spectrometry. Sample preparation, sample analysis, and data processing are fully automated using Biomek ADME-TOX workstation and the UV-based PAMPA Evolution-96 Command Software (Beckman Coulter, Inc.; Fullerton, CA) .

EXAMPLE 7 : Cytotoxicity

[00038] The activity of compounds against the Caco2 cell line was determined as previously described in 10% FBS

(Hurdle, J.G. et al . 2009. Anti icrob. Agents Chemother. 53 :4028-4031) .

Claims

What is claimed is:

1. A method for killing Clostridium difficile organisms comprising contacting Clostridium difficile organisms with reutericyclin or a reutericyclin analog compound .

2. The method of claim 1 wherein said Clostridium difficile organisms are log phase or stationary phase organisms .

3. A method for treating Clostridium difficile- associated diarrhea which comprises administering to a patient with Clostridium difficile-associated diarrhea an effective amount of reutericyclin or a reutericyclin analog compound in a pharmaceutically acceptable vehicle.

4. A method for preventing relapse of C. difficile infection which comprises administering to a patient with Clostridium difficile infection an effective amount of reutericyclin or a reutericyclin analog compound in a pharmaceutically acceptable vehicle so that relapse of C. difficile infection is prevented.

5. The method of claim 3 wherein said patient is administered an effective amount of reutericyclin or a reutericyclin analog in combination with an anti-infective agent .

6. The method of claim 4 wherein said patient is administered an effective amount of reutericyclin or a reutericyclin analog in combination with an anti - infective agent .