Compounds and methods for improving health

Abstract

The present technical field relates to methods for promoting gut microbiome health using low G+C preference (LDN) analogs. More specifically, the technical field relates to methods for selectively reducing physiologically harmful Gram-positive bacteria with genomes low in guanine and cytosine (rather than genomes containing higher amounts of adenine and thymine / uracil nucleotides) in the gut microbiome, while maintaining or increasing the proportion of beneficial microflora, using LDN analogs as inhibitors of the DNA polymerase IIIC (DNA pol IIIC) enzyme.

Description

[Technical Field]

[0001] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0002] The present technical field relates to methods for promoting gut microbiome health using low G+C preference (LDN) analogs. More specifically, the technical field relates to methods for selectively reducing physiologically harmful Gram-positive bacteria with genomes low in guanine and cytosine (rather than genomes containing higher amounts of adenine and thymine / uracil nucleotides) in the gut microbiome, while maintaining or increasing the proportion of beneficial microflora, using LDN analogs as inhibitors of the DNA polymerase IIIC (DNA pol IIIC) enzyme. [Background technology]

[0003] The mucosal surfaces of the body contain complex and specialized microbial communities, often referred to as the microbiome or microbiota (Mullish BH, Quraishi MN, Segal JP, et al., The gut microbiome: what every gastroenterologist needs to know, Frontline Gastroenterology 2021;12:118-127). The human gastrointestinal microbiome is estimated to consist of up to 100 trillion microorganisms, most of which are found in the large intestine (Kachrimanidou M, Tsintarakis E. Insights into the Role of Human Gut Microbiota in Clostridioides difficile Infection. Microorganisms. 2020;8(2):200 https: / / doi.org / 10.3390 / microorganisms8020200). The gut microbiome is diverse, but in healthy adults, it is primarily composed of bacteria from two major phyla: Firmicutes (Gram-positive, spore-forming organisms) and Bacteroidetes (Gram-negative, non-spore-forming organisms). These two phyla typically comprise approximately 90% of the microbiome (Mullish BH, Quraishi MN, Segal JP, et al., The gut microbiome: what every gastroenterologist needs to know, Frontline Gastroenterology 2021;12:118-127).

[0004] In addition to the Firmicutes and Bacteroidetes phyla, the gut microbiome is also composed of additional bacteria, including Actinobacteria, Fusobacteria, Verrucomicrobia, and Proteobacteria (Mullish BH, Quraishi MN, Segal JP, et al., The gut microbiome: what every gastroenterologist needs to know, Frontline Gastroenterology 2021;12:118-127). The Proteobacteria phylum is composed of gram-negative facultative anaerobes. Although some members of the phylum Proteobacteria are part of a healthy gut in low abundance, this phylum also includes common unwanted Gram-negative pathogenic commensals such as Salmonella, Shigella, and Escherichia coli (Mullish BH, Quraishi MN, Segal JP, et al., The gut microbiome: what every gastroenterologist needs to know, Frontline Gastroenterology 2021;12:118-127).

[0005] Actinobacteria are present in large proportions in children and generally decline in overall proportion with age (replaced by Firmicutes and Bacteroidetes). At birth, facultative anaerobic species such as E. coli, Staphylococcus, and Streptococcus colonize the infant gut, creating an anaerobic environment within the first few days of life that allows strict anaerobes (anaerobes that cannot grow in the presence of more than 5 μM dissolved oxygen) such as Bacteroides (phylum Bacteroidetes) and Bifidobacterium (phylum Actinobacteria) to thrive (Mueller NT, et al., The infant microbiome development: mom matters. Trends Mol Med. 2015 Feb;21(2):109-17. doi:10.1016 / j.molmed.2014.12.002. Epub 2014 Dec 11. PMID: 25578246; PMCID: PMC4464665). Over the first year of life, infant exposure to the environment and breast milk or formula contributes to the evolution of the gut microbiome into a mature microbiome that resembles that of an adult. (Jangi, S. and Lamont, T., (2010) Asymptomatic Colonization by Clostridium difficile in Infants: Implications for Disease in Later Life, Journal of Pediatric Gastroenterology and Nutrition. 51(1):2-7).

[0006] The gut microbiome exhibits a symbiotic relationship with the host. Through this symbiotic relationship, the microbiome provides numerous benefits to the host, including shaping the intestinal and systemic immune system, maintaining a healthy intestinal epithelium, harvesting energy from food, and protecting against pathogens. (Mullish BH, Quraishi MN, Segal JP, et al., The gut microbiome: what every gastroenterologist needs to know, Frontline Gastroenterology 2021;12:118-127) When the microbiome composition shifts from its normal diversity, these beneficial physiological functions are disrupted, a condition known as dysbiosis. (Mullish BH, Quraishi MN, Segal JP, et al., The gut microbiome: what every gastroenterologist needs to know, Frontline Gastroenterology 2021;12:118-127) When the gut microbiome is in a state of dysbiosis, it contains fewer beneficial microorganisms (commensals) and more potentially harmful microorganisms (pathogenic commensals) (Mullish BH, Quraishi MN, Segal JP, et al., The gut microbiome: what every gastroenterologist needs to know, Frontline Gastroenterology 2021;12:118-127).

[0007] Important to health are anaerobic bacteria, i.e., bacteria that thrive in an oxygen-depleted atmosphere, such as those found in the intestinal environment. Gram-positive anaerobes, such as Lactobacilli, Bifidobacteria, and Eubacteria, as well as Gram-negative anaerobes, such as Bacteroides, represent "good" gut flora important to health. In contrast, the Gram-positive anaerobes Clostridioides difficile and Clostridioides perfringens represent pathogenic bacteria. In particular, Clostridioides difficile (C. diff.) is increasingly associated with patient illness, likely due to the reliance on antibiotics used to treat pathogenic bacterial infections.

[0008] Modern medicine has been shaped by the emergence and use of antibiotic drugs. The mid-20th century has even been called the "antibiotic age." In fact, infectious diseases were thought to have been eradicated by the end of the last century (Huemer, M. et al., Antibiotic resistance and persistence—Implications for human health and treatment perspectives, EMBO Rep. 2020 Dec 3;21(12):e51034). Antibiotics are essential for treating and curing a range of bacterial infections, but an increasing number of bacteria are becoming resistant to the increasing number of antibiotics currently in use. This widespread use of antibiotics has led to bacterial microorganisms exhibiting multidrug resistance (MDR) (Tanwar J, Das S, Fatima Z, Hameed S (2014) Multidrug resistance: an emerging crisis. Interdiscip Perspect Infect Dis 2014:541340). Antibiotic-resistant bacteria pose a major threat to modern medicine because new antibiotics are scarce and the prevalence of MDR bacteria, which cause treatment failure, is increasing (Spellberg B, Bartlett JG, Gilbert DN (2013) The future of antibiotics and resistance. N Engl J Med 368:299-302). As a result, bacterial pathogens pose a serious threat to public health.

[0009] Nosocomial pathogens and infections are acquired in hospitals and pose a significant threat to hospital staff and patients. Nosocomial pathogenic bacteria with increasing levels of multidrug resistance and virulence are referred to as ESKAPE pathogens (Huemer, M. et al., Antibiotic resistance and persistence—Implications for human health and treatment perspectives, EMBO Rep. 2020 Dec 3;21(12):e51034). ESKAPE represents the Gram-positive bacterial species Enterococcus faecium and S. aureus, as well as the Gram-negative bacteria K. pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter species. ESKAPE pathogens impose a significant burden on patients' health and healthcare systems. These pathogens are characterized by high levels of MDR and are responsible for causing nosocomial and potentially life-threatening infections in critically ill and immunocompromised patients (Huemer, M. et al., Antibiotic resistance and persistence—Implications for human health and treatment perspectives, EMBO Rep. 2020 Dec 3;21(12):e51034; and Rice LB (2010) Progress and challenges in implementing the research on ESKAPE pathogens. Infect Control Hosp Epidemiol 31(Suppl 1):S7-10).As microbial resistance to many antimicrobial drugs increases, the use of "last-resort" antibiotics, such as tigecycline, polymyxin E, daptomycin, vancomycin, and linezolid, has become more common (Li, W., et al., (2022), Evaluation of culturable 'last-resort' antibiotic-resistant pathogens in hospital wastewater and implications on the risks of nosocomial antimicrobial resistance prevalence. Journal of hazardous materials, 438, 129-477). These so-called last-resort antibiotics serve as the "last line of defense" against antibiotic-resistant pathogen infections. As a result, the increasing prevalence of "last-resort" antibiotic-resistant pathogens in hospital environments and their nosocomial transmission pose a serious threat to patient well-being (Li, W., et al., (2022), Evaluation of culturable 'last-resort' antibiotic-resistant pathogens in hospital wastewater and implications on the risks of nosocomial antimicrobial resistance prevalence. Journal of hazardous materials, 438, 129477).

[0010] Two Gram-positive pathogens, Staphylococcus aureus and Enterococcus fecalis / fecium, are primarily nosocomial pathogens and currently account for the majority of hospital-acquired illnesses. Furthermore, Clostridioides difficile infection (CDI) is the most common cause of healthcare-associated infections in the United States (Magill, et al., “Changes in Prevalence of Health Care-Associated Infections in US Hospitals,” The New England Journal of Medicine 2018, 379, 1732–1744). Importantly, C. difficile can be a normal component of a healthy gut microbiome. However, during periods of microbiome dysbiosis (which may be the result of antibiotic treatment), C. difficile can proliferate and cause disease, CDI.

[0011] Overgrowth of C. difficile in the intestinal environment can result in a wide range of clinical symptoms, from mild diarrhea to severe, life-threatening colonic perforation and toxic megacolon (extreme inflammation and distension of the colon). The use of last-resort antibiotics disrupts the host microbiome, allowing C. difficile to grow freely without competition (Davis ML, et al. Multicenter derivation and validation of a simple predictive index for healthcare-associated C. difficile infection. (Clin Microbiol Infect 2018;24:1190-4)). For example, treatment with broad-spectrum antibiotics can result in the near-complete loss of Bacteroidetes, a reduction in Firmicutes, and an overgrowth of Proteobacteria. These changes allow C. difficile spores to germinate, resulting in increased growth and increased pathogenic potential (Mullish BH, Quraishi MN, Segal JP, et al., The gut microbiome: what every gastroenterologist needs to know, Frontline Gastroenterology 2021;12:118-127). Despite the risk of antimicrobial resistance and limited treatment options, antimicrobial therapy remains the first line of defense against pathogenic microbial infections. For example, antibiotic therapy remains the primary method of treatment for pathogens such as S. aureus, C. difficile, E. faecalis / fecium, and other known bacterial pathogens.

[0012] In contrast to Gram-negative bacteria, which are poorly permeable to many antimicrobial compounds (e.g., vancomycin), the Gram-positive pathogenic commensal organism S. aureus (S. aureus) is naturally susceptible to nearly all antibiotics developed. However, despite antimicrobial susceptibility, S. aureus and similar pathogenic commensals are known to rapidly acquire antibiotic resistance. In many cases, such pathogenic commensals acquire antimicrobial resistance by acquiring specific genetic modifications, such as advantageous mutations or by undergoing horizontal gene transfer. As a result of the back-and-forth relationship between antimicrobial development and the evolution of pathogenic commensals, these types of pathogenic bacterial infections tend to occur in epidemic waves (Chambers HF, Deleo FR (2009) Waves of resistance: Staphylococcus aureus in the antibiotic era. Nat Rev Microbiol 7:629-641). For example, vancomycin is recommended by the Infectious Disease Society of America (IDSA) treatment guidelines, but it has been associated with high rates of CDI recurrence and increased resistance due to disruption of the host microbiota (Isaac S, et al., Short- and long-term effects of oral vancomycin on the human intestinal microbiota. J Antimicrob Chemother 2017;72:128-36; and Peng Z, et al., Update on antimicrobial resistance in C. difficile: resistance mechanisms and antimicrobial susceptibility testing. (J Clin Microbiol 2017;55:1998-2008)).

[0013] Furthermore, treatment with antibacterial compounds such as vancomycin can reduce microbiome diversity in the phyla Firmicutes, Actinobacteria, and Bacteroidetes, along with characteristic Proteobacteria overgrowth (Garey KW, et al. A randomized, double-blind, placebo-controlled, single- and multiple-ascending-dose Phase 1 study to determine the safety, pharmacokinetics, and food and faecal microbiome effects of ibezapolstat administered orally to healthy subjects. J Antimicrob Chemother 2020;75(12):3635-3643). Proteobacteria overgrowth is associated with a significantly increased risk of systemic infections with MDR Gram-negative microorganisms. Vancomycin for the treatment of enteric bacterial infections is associated with a high recurrence rate and dysbiosis, with approximately 20–25% of patients experiencing recurrent infections after discontinuing treatment. (Gonzales-Luna AJ, Carlson TJ, Dotson KM, et al. PCR ribotypes of Clostridioides difficile across Texas from 2011 to 2018, including emergence of ribotype 255. Emerg Microbes Infect 2020;9(1):341-7). Therefore, new therapeutic approaches with distinct mechanisms of action against harmful bacterial pathogenic commensals are urgently needed.

[0014] In particular, there is a need for compounds that can reduce or eliminate the overgrowth of harmful microorganisms in the intestinal environment, which can cause infection or disease, while maintaining the delicate balance of microorganisms that make up the intestinal microbiome. Such treatments can result in clinical cures and also provide protection against recurrent overgrowth, infection, and / or dysbiosis. Therefore, there is also a need for treatments that promote the health of the microbiome. Summary of the Invention

[0015] The methods and compositions provided herein relate to promoting gut microbiome health by administering low G+C-directed nucleoside (LDN) analogs to a subject.

[0016] Provided herein is a method for promoting the health of a subject's gut microbiome, comprising administering an effective amount of a low G+C-preferring nucleoside (LDN) analog to a subject suffering from intestinal dysbiosis, wherein the LDN analog maintains and / or increases beneficial microorganisms in the gut microbiome while simultaneously reducing or eliminating the growth of harmful Gram-positive bacteria with low G+C content in the gut microbiome. In some embodiments, the LDN analog can be a small molecule inhibitor of DNA pol IIIC enzyme, such as ibezapolstat. In some embodiments, the LDN analog can target DNA pol IIIC of low G+C class bacteria whose genomes contain less than 50% guanine (G) + cytosine (C). In some embodiments, LDN analogs can selectively target physiologically harmful species belonging to the genus Streptococcus, Enterococcus, Staphylococcus, Bacillus, Clostridioides, Pneumococcus, Listeria, Mycoplasma, and / or Lactobacillus. In some embodiments, beneficial microorganisms may include the phylum Firmicutes, which may include the families Lachnospiraceae and Lactobacillaceae. In yet other embodiments, administering an effective amount of an LDN analog can reduce or prevent the re-growth of low G+C content Gram-positive pathogenic commensals in the gut microbiome within 30 days.

[0017] Also disclosed herein are methods for achieving and / or maintaining a healthy proportion of gut microflora in a subject's gut environment, comprising administering to the subject an effective amount of a compound against the DNA pol IIIC enzyme in low G+C content Gram-positive pathogenic commensals, thereby reducing or eliminating physiologically harmful pathogenic microorganisms belonging to the phylum Bacillus, and increasing and / or maintaining physiologically beneficial microorganisms in the gut environment. In some examples, the compound may be an LDN analog. In some embodiments, administration of the LDN analog may be prophylactic, the subject may be healthy, and the LDN analog may restore or maintain a symbiotic relationship between the subject and the microorganisms in the gut environment. Alternatively or additionally, in some embodiments, the subject may be suffering from an overgrowth of Clostridioides difficile in the gut. In some instances, physiologically beneficial microorganisms in the intestinal environment include anaerobic gram-positive bacteria belonging to the genus Clostridium, and may include C. coccoides.

[0018] Also provided herein are compositions for promoting gut microbiome health, comprising low G+C-directed nucleoside (LDN) analogs, wherein the LDN analogs inhibit DNA pol IIIC enzymes in physiologically harmful pathogenic commensals, thereby reducing harmful Gram-positive bacteria while allowing beneficial microorganisms to flourish in the gut microbiome. Additionally, in some embodiments, the LDN analogs can be a preventative treatment, promoting the persistence and / or repopulation of a healthy microbiota. In some instances, LDN analogs may selectively reduce the growth of pathogenic members of the Streptococcus, Enterococcus, Staphylococcus, Bacillus, Clostridioides, Pneumococcus, Listeria, Mycoplasma, and / or Lactobacillus.

[0019] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application file with one or more color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0020] The drawings described herein are for illustration purposes only and are not intended to limit the scope of the present disclosure in any way. [Brief explanation of the drawings]

[0021] [Figure 1] Figure 1 presents the results of qPCR experiments: Figure 1A shows the quantification of relevant Firmicutes isolated from samples taken from healthy volunteers in Phase 1, and Figure 1B shows the quantification of relevant Firmicutes isolated from samples taken from subjects with CDI in Phase 2a. [Figure 2] Figure 2 is a schematic diagram showing Firmicutes Ibezapolstat (IBZ) susceptibility. Figure 2A shows variable susceptibility between Clostridium butyricum strains C. butyricum 1008 and C. butyricum 1007. Figure 2B is a depiction and comparison of the structures of the two C. butyricum strains. [Figure 3] Figure 3 is a graph showing the target flagellar genes on the x-axis and the relative expression of these genes compared to the untreated control on the y-axis. Additionally, to better visualize the data, the relative expression of the control was normalized to a value of 1. [Figure 4] FIG. 4 shows a motility assay comparing the motility of a control strain of C. difficile to a strain obtained from an isolate and exposed to a sub-inhibitory (but not killing) concentration of ivezapolstat. [Figure 5]Figure 5 is a graph showing changes in the gut microbiome after antibiotic administration in patients with C. difficile infection (CDI), illustrating how this may increase the likelihood of recurrent CDI. The graphic was designed using BioRender. [Figure 6] Figure 6 shows a graph illustrating the germ-free humanized mouse study design. The graphic was created using BioRender. [Figure 7] Figures 7A-E show boxplots with lines connecting groups based on color. The colors represent the antibiotics to which mice were exposed during the last 10 days of the experiment (or lack thereof, in the case of the no-drug control (ND control) and baseline samples). Figures 7A and 7B show an illustration of the change in alpha diversity of the gut microbiome throughout the experiment. Figure 7A shows the change in abundance of Operational Taxonomic Units (OTUs) within the gut microbiome over the course of the experiment. Figure 7B shows the change in inverse Simpson's index within the gut microbiome over the course of the experiment. Figures 7C, 7D, and 7E present an illustration of the change in beta diversity (diversity between groups) of the gut microbiome throughout the experiment. Figure 7C shows the change in gut microbiome beta dispersion (centroid distance) throughout the experiment; Figure 7D shows the change in Bray-Curtis dissimilarity relative to baseline 1 (D7) throughout the experiment; Figure 7E shows the change in Bray-Curtis dissimilarity relative to baseline 2 (D14); and Figure 7F shows non-metric multidimensional scaling (NMDS) of Bray-Curtis dissimilarity. Each dot represents one sample collected at each time point (labeled at the top of each plot). The size indicates the inverse Simpson's index value for that sample, the shape indicates which trial the sample was collected from, and the color indicates which antibiotic (or lack thereof) each sample was exposed to. [Figure 8]Figures 8A-D show stacked bar graphs showing the average relative abundance (expressed as percentages) of different bacterial taxonomic levels across the experiment: Figure 8A represents the phylum level, Figure 8B represents the class level, Figure 8C represents the order level, and Figure 8D represents the family level. [Figure 9] Figures 9A-D show bar graphs of OTUs identified by random forest analysis that distinguish specific treatment groups. The dashed line represents the significance cutoff based on median importance given one standard deviation in both directions, and the OTUs shown (x-axis) are categorized at the family level. The comparative and enriched (elevated) OTUs indicated in the legend are colored for each panel. Figures 9A-D show a comparison of each group of mice exposed to antibiotics or a no-drug control group (ND control). Figure 9E compares mice exposed to ibexapolstat with mice exposed to fidaxomicin. [Figure 10] FIG. 10 shows the effect of ibezapolstat on the morphology of C. difficile CD 630 strain. [Figure 11] FIG. 11 shows the effect of ibezazolstat on CD630 motility. [Figure 12] FIG. 12 shows the effect of ibezazolstat on flagellar gene expression. [Figure 13] FIG. 13 shows the effect of ibezapolstat on modulating C. difficile toxin production. [Figure 14] Figure 14 presents a basic flow chart outlining the experimental procedures used in these studies. [Figure 15] FIG. 15 shows biofilm formation by C. difficile laboratory strains R20291 and CD630. [Figure 16] FIG. 16 shows the minimum biofilm inhibitory concentration (MBIC) and Eagle effect in response to antibiotic treatment with IBZ, vancomycin (VAN), fidaxomicin (FDX) or metronidazole (MTZ). [Figure 17]FIG. 17 is a graph demonstrating the effect of MIC or sub-MIC levels of IBZ and VAN on early (4 hour) biofilms by measuring CFU / mL over a 24 hour or 48 hour period. [Figure 18] FIG. 18 is a graph demonstrating the effect of MIC or sub-MIC levels of IBZ and VAN on early (4 hour) biofilms by measuring optical density at A570 over a 24 hour or 48 hour period. [Figure 19] FIG. 19 shows the effect of IBZ and comparable antibiotics on early stage (24 hour) biofilms containing either C. difficile laboratory strains R20291 or CD630, as measured by optical density at A570 over 24 or 48 hours. DETAILED DESCRIPTION OF THE INVENTION

[0022] The features and other details of the present invention will now be described in more detail. It will be understood that the particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention.

[0023] Described herein are methods and compositions for promoting gut microbiome health by administering low G+C-directed nucleoside (LDN) analogs, such as inhibitors of DNA pol IIIC enzymes, to a subject. The LDN analogs described herein can target bacteria of the low G+C class whose genomes contain less than 50% guanine (G) + cytosine (C). Furthermore, the provided LDN analogs can selectively inhibit physiologically harmful pathogenic symbionts belonging to the phylum Bacillus, which may include members of the phylum Firmicutes or the order Bacillales. Furthermore, the LDN analogs can inhibit the growth or replication of pathogenic symbionts of the genus Clostridium, such as C. difficile, while promoting the growth of beneficial members of the genus Clostridium (e.g., C. coccoides). In some examples, provided LDN analogs may selectively target gram-positive members of the Bacillales order, including members of the Staphylococcus family and / or Enterococcus, such as S. aureus and E. faecium. Furthermore, LDN analogs may simultaneously promote the persistence or repopulation of healthy microbiota in the intestinal environment. For example, the proliferation of Actinobacteria may be increased in a subject's intestinal microbiome upon administration of provided LDN analogs. In some embodiments, LDN analogs may selectively target at least one single nucleotide polymorphism (SNP) in healthy microbiota organisms, allowing for the continued growth of these healthy organisms in the presence of LDN.

[0024] Also described herein are methods and compositions that can target flagellar genes and thus reduce the motility of Gram-positive organisms.For example, provided compositions can reduce flagellar gene expression, including common flagella-related genes fliA, flgB, fliC-VIP.

[0025] Ibezapolstat (IBZ) is a non-absorbable antibacterial agent for the treatment of C. difficile infection (CDI). In vitro and human studies have demonstrated potent activity of IBZ against C. difficile, but also selective activity against other beneficial Gram-positive gut microbiota, which has been shown to reduce the risk of dysbiosis. While the target DNA pol IIIC enzyme is present in most Gram-positive species, IBZ susceptibility demonstrates selectivity among certain Gram-positive species that may be found in the gut microbiota.

[0026] definition A low G+C-specific nucleoside (LDN) analog refers to any molecule and / or compound of the class of nucleoside analog inhibitors that can selectively target and inhibit the DNA polymerase IIIC (DNA pol IIIC) enzyme of Gram-positive microorganisms (bacteria with genomes containing fewer guanine and cytosine nucleotide bases as opposed to adenine and thymine / uracil bases) with a low G+C content, such as the Firmicutes and Bacillales, which have a low G+C content. Such molecules may include, but are not limited to, 7-substituted-N2-(3,4-dichlorobenzyl)guanine (DCBG), which can selectively inhibit DNA pol IIIC. Such molecules may also be 1,7-dihydro-6H-purin-6-one compounds, such as those described in U.S. Patent Nos. 6,926,763 and 8,796,292, which are incorporated herein by reference.

[0027] For example, ibezaporstat is 2-((3,4-dichlorobenzyl)amino)-7-(2-morpholinoethyl)-1,7-dihydro-6H-purin-6-one.

[0028] An "effective amount" refers to an amount sufficient to produce a beneficial or desired clinical or biochemical result. An effective amount can be administered one or more times. For example, an effective amount can be an amount that, when administered to a site of infection or potential infection, treats or prevents bacterial infection while simultaneously increasing the amount and / or proportion of Actinobacteria and / or Firmicutes in the microbiome.

[0029] "Selective target" or "selective targeting" refers to a mechanism that is specifically targeted to inhibit DNA pol IIIC enzymes present in Gram-positive microorganisms that have a genome low in G+C (having fewer guanine and cytosine nucleotides based on their genome than adenine and thymine / uracil bases).

[0030] "Administration" or "administering" refers to a method (e.g., topical, oral, intravenous, intraperitoneal, or intramuscular) of providing one or more unit doses of an LDN analog, such as ibezazolstat, to an animal, e.g., a mammal. The method of administration can vary depending on various factors, such as the components of the pharmaceutical composition, the site of potential or actual infection, and the severity of the actual microbial infection.

[0031] By "inhibit" is meant reducing the rate of bacterial cell growth by at least 80%. In certain embodiments, growth may be inhibited by 90%, 95%, or even 99% or more. The degree of inhibition may be confirmed, for example, by an in vitro growth assay, for example, by standard liquid culture techniques. Inhibition of colony formation at a suitable MIC (minimum inhibitory concentration), for example, <100 μg / ml, more preferably <10 μg / ml, is preferred.

[0032] "Treatment" refers to an approach for obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delay in disease progression, improvement or palliation of the disease state, and remission (whether partial or total). "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder, as well as those in whom the disorder is to be prevented and / or recurrence is to be prevented. "Alleviating" a disease means that the severity of the disease state and / or undesirable clinical symptoms are reduced and / or the time course of progression is slowed or prolonged compared to the situation without treatment.

[0033] "Microbiome" refers to the microorganisms in a particular environment (including the body or part of the body). Preferably, the microbiome is located in the gut.

[0034] "Gut environment" refers to the internal environment of the intestinal system, which may harbor the gut microbiota, including the complex community of bacteria, archaea, fungi, viruses, and protozoa that provide the host organism with a supply of cells and genes in greater numbers than itself.

[0035] A "healthy microbiome" can be described in terms of ecological stability (i.e., the ability to resist changes in community structure under stress or to rapidly return to baseline after stress-related changes) with an idealized (presumably health-related) composition or with a desirable functional profile (including metabolic and nutrient supply to the host). The microbiome of a healthy adult may also be characterized by a majority of bacterial species in the Firmicutes or Bacteroidetes phyla, and a minority in the Actinobacteria and Proteobacteria phyla. The microbiome of a healthy newborn may be characterized by a majority of bacterial species in the Bacteroidetes and Actinobacteria phyla.

[0036] "Improving gut microbiome health" means that the microbiome is composed of a majority of bacterial species from the phyla Actinobacteria, Firmicutes, or Bacteroidetes, with a minority of Proteobacteria. Alternatively, improving gut microbiome health can mean increasing the proportion of bacterial species from the phylum Actinobacteria, as present in the gut microbiome of a healthy newborn. The subject may or may not be suffering from a pathogenic bacterial infection.

[0037] "Reducing the likelihood of infection" means a prophylactic treatment or treatment that results in a reduction (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) in the likelihood or rate of a subject or patient population developing a microbial infection by administering the compound compared to a subject or patient population not receiving the LDN analog.

[0038] "Clinical cure" means that the initial infection has resolved, preferably as measured after the subject has received treatment, approximately 10-12 days after diagnosis.

[0039] "Sustained clinical cure" means that the subject has had a clinical cure and has not had a recurrence of infection, as measured 30-90 days after diagnosis.

[0040] By "relapse" is meant that the subject has had a clinical cure and the infection reoccurs within 30 to 90 days.

[0041] "Animal" refers to any animal susceptible to gram-positive bacterial infection. Such animals may include humans, dogs, cats, pigs, cows, horses, goats, chickens, turkeys, sheep, rats, mice, and rabbits, as well as other animals kept commercially or as pets. "Animals susceptible to microbial infections" are defined as animals that are at higher risk of contracting microbial infections compared to the general population. Examples of such animals include animals that have recently undergone surgical procedures, or immunocompromised humans, such as animals with AIDS (acquired immune deficiency syndrome), or animals with transplants that require immunosuppressive drugs. Such animals can be identified using methods known to those skilled in the art.

[0042] A "coating agent" is defined as a biocompatible compound or mixture of compounds suitable for coating a surface. Suitable coating agents are known in the art. Exemplary coating agents include, but are not limited to, polymers such as polyethylene glycol, hypromellose, hydroxypropyl cellulose, polytetrafluoroethylene, methylcellulose, polyvinyl alcohol, or other biocompatible polymers.

[0043] A "media" is defined as any substance, liquid, or solid on or in which microorganisms can exist or in which it is desired to prevent the presence of microorganisms. Exemplary media include media (e.g., agar or broth), food, medical supplies (e.g., sterile fluids), medical devices (e.g., catheters), countertops, and other surfaces.

[0044] A "microbial infection" is defined as the invasion of a host animal by a pathogenic microorganism. For example, an infection can include the overgrowth of a microorganism normally present in or on an animal, or the growth of a microorganism not normally present in or on an animal. More generally, a microbial infection can be any situation in which the presence of a microbial population(s) is damaging to a host animal. Thus, an animal is "suffering" from a microbial infection when an excessive amount of a microbial population is present in or on an animal, or when the presence of a microbial population(s) is damaging to the animal's cells or other tissues. In one embodiment, the number of a particular genus or species of microorganism is at least 2, 4, 6, or 8 times the number normally found in the animal. Examples of microorganisms include, but are not limited to, gram-positive or any other class of bacteria.

[0045] "Pharmaceutically acceptable salts" refers to salts derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Other acids, such as oxalic acid, are not pharmaceutically acceptable per se, but may be useful as intermediates in obtaining the compounds of the present invention and their pharmaceutically acceptable acid addition salts. Salts derived from suitable bases include alkali metal (e.g., sodium or potassium), alkaline earth metal (e.g., magnesium), ammonium, and NR4 + (Wherein R is C 1~4Preferred salts include the hydrochloride, hydrobromide, sulfate, mesylate, maleate, tartrate, and fumarate salts. References hereinafter to compounds according to the invention include compounds of the general formula shown and their pharmaceutically acceptable salts.

[0046] "Prevention" of microbial growth or infection is defined as application of a compound of the invention such that microbial growth or infection does not occur. The amount of a compound of the invention required to prevent microbial growth can be determined, for example, by an in vitro growth assay, e.g., by standard liquid culture techniques. The amount of a compound of the invention required to prevent microbial infection can be determined, for example, by an in vivo assay, e.g., by determining the amount of compound that must be administered to prevent infection in a research animal, e.g., a guinea pig, after inoculation with the microorganism. Generally, compounds that demonstrate prevention at appropriate concentrations, e.g., <100 μg / ml, more preferably <10 μg / ml, are useful for further testing as therapeutic agents.

[0047] "Treating" is defined as the medical management of a patient with the intent to result in a cure, amelioration, or prevention of a disease, pathological condition, or disorder. The term includes active treatment, i.e., treatment specifically directed at ameliorating a disease, pathological condition, or disorder, and also includes causal treatment, i.e., treatment directed at eliminating the cause of a disease, pathological condition, or disorder. In addition, the term includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure a disease, pathological condition, or disorder; prophylactic treatment, i.e., treatment directed at preventing a disease, pathological condition, or disorder; and supportive treatment, i.e., treatment used to supplement another specific therapy aimed at ameliorating a disease, pathological condition, or disorder. The term "treating" also includes symptomatic treatment, i.e., treatment directed at the essential symptoms of a disease, pathological condition, or disorder.

[0048] A "therapeutically effective amount" is defined as an amount that, when administered to an animal in need thereof, alleviates at least some of the symptoms of a bacterial infection. In the context of prophylaxis, a "therapeutically effective amount" is an amount that, when administered to an animal susceptible to a bacterial infection, serves to inhibit or otherwise reduce the likelihood of such infection.

[0049] "Prophylactic" or "prophylactic treatment" refers to administering an effective amount of an LDN analog to a subject who may be considered healthy, i.e., the subject does not exhibit signs or symptoms of disease or dysbiosis in the intestine. The subject may or may not be susceptible to intestinal dysbiosis and / or infection. For example, an LDN analog may be administered to a subject to maintain a healthy intestinal microbial balance, promote a symbiotic relationship between the host intestine and the microorganisms therein, and / or reduce the growth, replication, or proliferation of physiologically harmful pathogenic commensals so that physiologically beneficial bacteria can thrive.

[0050] The details of one or more embodiments of the invention are set forth in the accompanying description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.

[0051] A healthy gut microbiome is composed of major bacterial groups called phyla. Firmicutes (Gram-positive spore-forming organisms) and Bacteroidetes (Gram-negative non-spore-forming organisms) are the most common, typically comprising over 90% of the gut microbiome in healthy adults. The adult gut microbiome also contains Actinobacteria, Fusobacteria, Verrucomicrobia, and Proteobacteria. Actinobacteria are overrepresented in children and generally decline in overall proportion with age (being replaced by Firmicutes and Actinobacteria). Proteobacteria (Gram-negative facultative anaerobes) typically comprise 2–5% of the healthy microbiome. When the composition of the microbiome changes from its normal diversity, normal physiological function is disrupted (called dysbiosis).Patients suffering from bacterial infection, such as C. difficile infection, are in a state of dysbiosis.Typically, the dysbiosis associated with subjects suffering from bacterial infection includes an increase in the proportion of Proteobacteria (often called "bloom") and a decrease in the number of Firmicutes and Bacteroidetes.

[0052] Certain bacteria, such as those of the Firmicutes phylum, have been found to be uniquely and selectively sensitive to inhibitors of DNA pol IIIC enzymes. Accordingly, 1,7-dihydro-6H-purin-6-one compounds and methods for synthesizing these compounds, as well as their use in inhibiting bacterial growth, are disclosed in U.S. Patent Nos. 6,926,763 and 8,796,292, which are incorporated herein by reference. For example, ibezaporstat is 2-((3,4-dichlorobenzyl)amino)-7-(2-morpholinoethyl)-1,7-dihydro-6H-purin-6-one. For example, ibezaporstat exhibits antibacterial activity against a range of Gram-positive bacteria and has been shown to be useful in treating C. difficile infections. Generally, the mechanism of action of DNA pol IIIC inhibitors targets Gram-positive bacteria (e.g., Firmicutes and Bacillales) with low G+C (fewer G and C DNA bases than A and T bases) content. The DNA polymerase IIIC (DNA pol IIIC) enzyme is essential for replication in low-G+C content Gram-positive pathogenic symbionts (microorganisms with genomes containing fewer guanine and cytosine bases than adenine and thymine / uracil bases). Therefore, LDN analogs targeting DNA pol IIIC may be selective or may selectively target Gram-positive pathogenic symbionts with low G+C genome content. At the same time, such LDN analogs may be inactive against other host microbiota, such as Actinobacteria or Bacteroidetes. In other words, Gram-positive bacteria containing low G+C genome content may be more susceptible to LDN analog treatment, while other bacteria (beneficial Firmicutes, Actinobacteria, Bacteroidetes and / or Gram-negative bacteria) may not be susceptible to LDN analog treatment.The majority of Gram-positive bacteria require the DNA pol IIIC enzyme for replication, yet susceptibility to DNA pol IIIC inhibitors varies among strains. Furthermore, susceptibility to small molecule DNA pol IIIC inhibitors (e.g., LDN analogs) was found to vary among the beneficial strains of the Firmicutes tested. Surprisingly, susceptibility not only varied among heterologous groups of different species, but also among isolates belonging to the same species (Table 4). As seen in Table 1, compared to the reference strain of Clostridium butyricum, one SNP was identified between the susceptible strain C. butyricum 1008, Y240D (Tyr240Asp), and two SNPs were identified between the less susceptible strain C. butyricum 1007, Y38D and D146E (Tyr38Asp and Asp146Glu). These results suggest that small molecule inhibitors of DNA pol IIIC can be designed to target these aspects and provide improved selectivity against low-G+C Gram-positive pathogenic commensals. Furthermore, inhibitors such as the provided LDN analogs may circumvent antibiotic resistance.

[0053] The present disclosure may provide methods for improving gut microbiome health. The methods and compositions provided herein may simultaneously reduce the population of physiologically harmful low G+C content Gram-positive pathogenic commensals in a subject's gut microbiome and promote the persistence, enhanced growth, and / or regrowth of physiologically beneficial gut microbiota.

[0054] In some embodiments, the disclosed LDN analogs can affect flagellar genes of some bacteria, such as fliA, flgB, and fliC-VIP, resulting in reduced motility of these organisms. The bacteria can be Gram-positive, including, but not limited to, C. difficile.

[0055] Some bacterial species possess a flagellum, and some species possess multiple flagella, or flagella. Flagella can be considered a motile organelle that enables movement and chemotaxis; however, flagella vary among bacterial species and may exhibit other functions that change during the bacterial life cycle. Furthermore, bacterial motility can play an important role in bacterial colonization and survival. For example, flagella enable bacteria to swim and swarm, thus providing advantages to such bacterial species in accessing nutrients, evading natural enemies, and colonizing new environments. Furthermore, flagella have been shown to be involved in regulating protein transport, host cell adhesion, cell invasion, autoaggregation, colony formation, and biofilm formation, and may also be involved in the secretion of non-flagellated bacterial proteins that may be involved in pathogenic processes. Different bacterial species use different mechanisms for motility, including twitching, swimming, gliding, and turning. In addition to flagella, motility can also involve the use of pili and other appendages, which can be influenced by environmental factors and the physical and chemical properties of the surface. For example, some bacterial species can use motility to transmit and form biofilms (Harshey RM (2003). Bacterial motility on a surface: many ways to a common goal. Annual review of microbiology, 57, 249-273). Consequently, flagella can strongly influence the virulence of bacterial pathogens and, therefore, may play an important role in bacterial pathogenesis.

[0056] Some examples of Gram-positive bacterial species that have at least one flagellum include Listeria monocytogenes, Clostridium botulinum, Clostridium tyrobutyricum, Staphylococcus aureus, Bacillus subtilis, and C. difficile.

[0057] Bacterial flagella may be positioned in one location, providing forward movement (polarity), or several flagella may spread across the bacterial surface (periphery), allowing them to roll in place. A flagellum is a long, whip-like appendage that protrudes from the surface of motile bacteria and rotates to propel the organism. A flagellum contains three main parts: a filament containing the protein flagellin (Flg), which acts as a propeller for bacterial motility; a basal body containing proteins involved in filament rotation; and a hook that transmits motor torque to the filament. The hook attaches the filament to the basal body and is composed of the hook protein FlgE. The filament may be a long, helical structure composed of flagellin FlgA and attached to the hook protein. The basal body is the motor that drives filament rotation and is composed of several proteins, including FliG, FliF, and FliM, while the flagellar basal body rod may contain FlgB and FlgC. Furthermore, the flagellar chaperone protein, FlgA, may play an important role in early flagellar development (Duan, Q., et al., Flagella and bacterial pathogenicity. Journal of basic microbiology, 53(1), 1-8(2013).). For example, regulation of C. difficile motility may occur, at least in part, at the level of flagellar gene expression.

[0058] Among other virulence factors, C. difficile expresses perifigella and toxins, which together promote diarrheal disease symptoms, pathology, and inflammation. Specifically, C. difficile flagella can promote bacterial motility and adherence to intestinal tissue, aiding in intestinal colonization, a necessary prerequisite for diarrheal disease symptoms. Furthermore, adherence levels of flagellated strains to the mouse cecum were observed to be 10-fold higher than those of non-flagellated strains (Tasteyre, A., et al., Role of FliC and FliD flagellar proteins of Clostridium difficile in adherence and gut colonization. Infect. Immun. 69:7937-7940 (2001)). It has also been suggested that flagellum function and regulation may contribute to the differences in permeability and virulence observed between different C. difficile strains. (Twine SM et al. Motility and flagellar glycosylation in Clostridium difficile. J. Bacteriol. 191:7050-7062 (2009).) Recent studies have also revealed that C. difficile, and possibly other Gram-positive anaerobic bacteria, may have the ability to phase-shift their virulence factors (i.e., switch between different phenotypes in response to environmental changes) to survive in different environments. Thus, the virulence of C. difficile, including the development of antibiotic resistance, may be one result of phase variation, and such phase variation may be influenced by the expression of flagellar genes (Anjuwon-Foster, BR, & Tamayo, R., Phase variation of Clostridium difficile virulence factors, Gut Microbes, 9:1, 76-83 (2018).)

[0059] Therapeutic Administration of Compounds In some embodiments, the LDN analogs provided herein can selectively inhibit the growth of harmful Firmicutes, such as C. difficile. Additionally, the provided compositions and methods can inhibit the growth of Bacillales, including members of the Staphylococcus family, such as S. aureus. Thus, the methods and compositions described in this disclosure can promote a healthy and balanced gut microbiome while reducing or eliminating harmful pathogenic commensals, which may include MDR strains of low-G+C Gram-positive pathogenic commensals, such as C. difficile, S. aureus, drug-resistant S. pneumoniae, and / or Enterococcus faecium. Furthermore, the provided LDN analogs can effectively treat other infectious diseases, including hospital-acquired infections. In some embodiments, the compositions and methods described herein may reduce or prevent the likelihood of recurrence of infection or dysbiosis caused by low G+C Gram-positive pathogenic commensals, including C. difficile infection.

[0060] Furthermore, the methods and compositions provided herein may improve the health of the gut microbiome through administration of an effective amount of an LDN analog. The subject need not be suffering from dysbiosis or an infection. The proportion of bacterial phyla in the subject's gut microbiome may be adjusted to a healthy balance compared to the person's gut microbiome before administration of the LDN analog. For example, the percentage of Actinobacteria may increase by approximately 5-50% upon exposure to an LDN analog.

[0061] In some aspects, in patients with C. difficile infection or dysbiosis, LDN analogs cause an overgrowth of healthy gut microbiota, such as species of the phyla Actinobacteria and Firmicutes, and an increase in the proportion of healthy microbiota, such as Clostridiales taxa, during and after treatment. In further embodiments, in patients with C. difficile infection or dysbiosis, LDN analogs initially increase the abundance of Actinobacteria, followed by a decrease in the abundance of Bacteroidetes and an increase in the abundance of Lachnospiraceae and Ruminococcaceae within 2-3 days of initiating treatment. In still further embodiments, overgrowth of healthy gut microbiota with treatment with LDN analogs results in 100% clinical cure at day 12 and 100% sustained clinical cure at day 38. For example, CDI can be completely eliminated with no recurrence of infection and an acceptable adverse event profile.

[0062] In some embodiments, treatment with LDN analogs may attenuate bacterial infection by targeting one or more virulence factors. In some embodiments, virulence factors may be selectively targeted to reduce harmful pathogenic commensals in the gut microbiome without affecting healthy microflora. For example, LDN analogs may inhibit flagellar gene expression in several Gram-positive organisms, including, but not limited to, C. difficile. In some examples, treatment of CDI with LDN analogs may reduce total toxin A and / or B production. By targeting bacterial virulence factors, LDN analogs may reduce overall bacterial fitness. For example, LDN analogs may modulate bacterial morphology and impair cell division. In some embodiments, LDN analogs may attenuate or eliminate CDI by inhibiting biofilm formation. For example, LDN analogs may reduce the biomass of C. difficile-embedded biofilms or biofilms containing Clostridium difficile.

[0063] In some embodiments, in patients not experiencing C. difficile infection or dysbiosis, LDN analogs induce an increase in the abundance of Actinobacteria, primarily Bifidobacteriales or Coriobacteriales, during or after administration. Regardless of the presence or absence of C. difficile infection or dysbiosis, LDN analogs preserved the proportion of Lachnospiraceae and the abundance of Clostridiales. In some embodiments, LDN analogs improve gut microbiome health by improving gut homeostasis through increasing the amount of C. coccoides. In some further embodiments, LDN analogs can stimulate the immune system to reduce inflammation and allergic diseases, as well as cellular components and metabolites such as butyrate, secondary bile acids, and indolepropionic acid. For example, LDN analogs can support the growth of C. coccoides in the gut microbiome, thereby enabling C. coccoides to produce short-chain fatty acids (SCFAs), which can inhibit the production of pro-inflammatory cytokines.

[0064] Alternatively, or in addition, LDN analogs may support the growth of bacterial species that produce anti-inflammatory cytokines. In some further embodiments, LDN analogs are used as probiotics to activate intestinal epithelial cells and strengthen the intestinal barrier. In further aspects, LDN analogs are used prophylactically to prevent, minimize, or reduce dysbiosis.

[0065] LDN analogs may meet the essential criteria for an ideal antibiotic for use against antibiotic-resistant bacterial pathogens that cause many hospital-acquired infections. For example, the DNA pol IIIC inhibitor ivezapolstat achieves high colonic concentrations with minimal systemic absorption, has potent activity against C. difficile while minimizing disruption of the gut microbiome, and is well tolerated in healthy volunteers and CDI patients. Alternatively or additionally, LDN analogs may be used as prophylactic agents against dysbiosis.

[0066] According to the methods of the present invention, the LDN analogs provided herein can be administered to a subject or patient in various forms depending on the selected administration route, as will be understood by those skilled in the art. For human or animal use, the LDN analogs can be administered orally, bucally, rectally, and vaginally, or topically, and can be administered as pharmaceutical compositions formulated accordingly. Preferably, the LDN analogs are administered in oral dosage form. For oral administration, the compositions can be in the form of, for example, tablets, capsules, granules, liquid solutions, and suspensions, but are not limited thereto. The compositions can also be administered by suppository or enema. For human or animal use, the formulations of the present invention can be administered parenterally, for example, intravenously, subcutaneously, intramuscularly, intraorbitally, ocularly, intraventricularly, intracranially, intracapsularly, intraspinally, intracapsularly, or intraperitoneally, or by intranasal, aerosol, cicatrization, oral, buccal, rectal, vaginal, or topical administration. The formulations of the present invention can also be administered as a bolus or by using a surgical implant that slowly releases the compound of the present invention over a preselected period of time.

[0067] As described above, LDN analogs can be administered to animals, preferably humans, alone or in combination with pharmaceutically acceptable excipients, with the proportion determined by the solubility and chemical properties of the compound, the selected route of administration, and standard pharmaceutical practice. LDN analogs can be administered to adults or children. The dosage of the compounds of the present invention and / or compositions containing the compounds of the present invention can vary depending on many factors, such as the mode of administration; the recipient's age, health, and weight; the nature and severity of symptoms; the frequency of treatment, and the type of concomitant treatment, if any; and the clearance rate of the compound in the treated animal. Those skilled in the art can determine the appropriate dosage based on the above factors. The compounds of the present invention can be initially administered at a suitable dosage, which can be adjusted as necessary depending on the clinical response. Generally, the compounds of the present invention can be provided in a physiologically buffered aqueous solution containing about 0.1-10% w / v of the compound, or in solid dosage forms such as tablets or capsules. Typical dosage ranges are about 0.01 mg / kg body weight to about 1 g / kg body weight per day. Oral dosages of ibezapolstat can include amounts of about 10 mg to 1000 mg per day, preferably 100 mg to 900 mg per day, and more preferably about 150, 300, 600, or 900 mg per day.

[0068] The LDN analogs can be formulated into pharmaceutical compositions for administration to human or animal subjects in a biologically compatible form suitable for in vivo or in vitro administration. Accordingly, the present disclosure provides pharmaceutical compositions comprising a compound of the present invention in admixture with an excipient.

[0069] The compounds described herein are useful for treating human intestinal microbial infections caused by Gram-positive bacteria, including strains resistant to common antibiotics. They are also useful for treating related Gram-positive bacterial infections in animals such as pigs, cattle, horses, goats, chickens, turkeys, sheep, rats, mice, and rabbits, as well as for eliminating or preventing bacterial or mycoplasmal infections in eukaryotic cell cultures or other media, such as food, cosmetics, medical devices, and hospital supplies.

[0070] The compounds of the present invention can be formulated with an acceptable diluent, carrier, or excipient, and / or in unit dosage form, for use in pharmaceuticals, veterinary medicine, and tissue culture. When using the compounds of the present invention, conventional pharmaceutical, veterinary, or tissue culture practices can be used to provide suitable formulations or compositions, all of which are encompassed by the pharmaceutical compositions of the present invention.

[0071] Non-limiting examples of parenteral formulations include liquid solutions or suspensions for oral administration, such as tablets, capsules, liquid solutions, and suspensions (e.g., solutions and suspensions intended for pediatric use), and intranasal formulations, such as powders, nasal drops, or aerosols. Other suitable formulations for parenteral, oral, or intranasal delivery of the compounds of the present invention are well known to those skilled in the art. Methods for preparing formulations well known in the art can be found, for example, in "Remington's Pharmaceutical Sciences." Formulations for parenteral administration may contain, as excipients, sterile water or saline, ethanol, polyalkylene glycols such as propylene glycol, polyethylene glycol, oils of vegetable origin, hydrogenated naphthalene, or biocompatible, biodegradable lactide polymers. Polyoxyethylene-polyoxypropylene copolymers can be used to control the release of the compounds of the present invention. Other potentially useful parenteral delivery systems for the compounds of the present invention include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain lactose as an excipient, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate, and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or may be gels for intranasal application. Formulations for parenteral administration may also contain glycocholate for buccal administration, methoxysalicylate for rectal administration, or citric acid for vaginal administration.

[0072] The concentration of the compound in the formulation of the present invention varies depending on several factors, including the administered dose and the route of administration. Generally, the compounds of the present invention can be provided in a physiologically buffered aqueous solution containing about 0.1 to 10% w / v of the compound for parenteral administration. Typical dose ranges are about 0.01 mg / kg to about 1 g / kg body weight per day, e.g., about 0.01 mg / kg to 100 mg / kg body weight per day. The administered dose depends on the type and progression of the infection being treated, the patient's overall health, and the route of administration. For topical and oral administration, formulations and dosages can be similar to those used for other antibiotics.

[0073] In one embodiment, the compound or composition of the present invention is administered to animals, such as humans, patients, who have been diagnosed with gram-positive bacterial infection.The compound can also be administered to animals or humans to inhibit or reduce the possibility of gram-positive bacterial infection, particularly in animals or humans that are susceptible to such infections (including but not limited to immunodeficient or immunocompromised human patients, or human patients who have recently undergone medical treatment).In other embodiments, cultured eukaryotic cells are treated with the new composition or the composition is added to inhibit or reduce the possibility of such infection (e.g., preventive treatment).The compound of the present invention can also be used to prevent bacterial growth in food, cosmetics, and pharmaceuticals, and on surfaces.

[0074] Compounds can be administered both prophylactically and after infection occurs.Prophylaxis may be most appropriate for immunocompromised human patients and animals, and for patients and animals after surgery or dental treatment.This list of relevant conditions for applying the method of the present invention is not intended to be limiting, and any suitable infection that responds to compound can be treated using the method and / or compound described herein.

[0075] Reduced transmissibility of infection In some aspects, the LDN analogs provided herein can be used to reduce the permeability of C. difficile or other Gram-positive pathogens, for example, by reducing bacterial cell motility. In some embodiments, LDN analogs can inhibit transmission by targeting one or more virulence factors, thus preventing the spread of harmful diseases. In some embodiments, LDN analogs can selectively inhibit virulence factors, such as flagellar genes, toxin production, and / or biofilm formation. For example, LDN analogs can reduce pathogenic transmission by inhibiting flagellar gene expression, thus reducing or eliminating bacterial motility. Furthermore, LDN analogs can modulate bacterial morphology and impair cell division. In some embodiments, surface treatment with LDN analogs reduces or inhibits biofilm formation. In some examples, LDN analogs can reduce the biomass of C. difficile-embedded biofilms or biofilms containing Clostridium difficile.

[0076] The compounds can also be used to treat or coat media or surfaces to prevent or reduce the extent of microbial growth. For example, the compounds of the present invention can be mixed with eukaryotic culture media (e.g., solid or liquid media) to prevent the growth of Gram-positive bacteria. Furthermore, the compounds of the present invention can be used in disinfectant formulations for treating surfaces, such as liquid formulations for cleaning and disinfecting surfaces in kitchens, bathrooms, hospitals, or other areas where medical procedures or potential microbial growth may occur. Medical devices and other surfaces can also be treated or coated with the compounds of the present invention to control microbial growth. Medical devices include those that are wholly or partially implanted in an animal and those that are external to the animal. Examples of medical devices include, but are not limited to, catheters, dialysis pumps, blood collection devices, stents, and drug delivery devices. Standard formulations for using the compounds of the present invention for surface treatment or coating are known to those skilled in the art.

[0077] example The foregoing description and examples have been set forth merely to illustrate the present invention and are not intended to be limiting. Since modifications of the described embodiments that incorporate the spirit and substance of the present invention may occur to those skilled in the art, the present invention should be broadly construed to include all modifications within the scope of the claims and equivalents thereof.

[0078] Example 1: DNA polymerase IIIC production The DNA polymerase IIIC-encoding gene can be amplified from genomic DNA. Primers can be designed to contain BamHI and XhoI cleavage sites at the 5' and 3' ends, respectively. The fragment can be directly inserted into the expression plasmid pET-28a(+) between the BamHI and XhoI sites. The recombinant protein can contain the full-length original protein as well as a His tag, a thrombin site, and a T7 tag at the N-terminus. The coding sequence of the recombinant protein can be confirmed by Sanger sequencing using eight primers covering the entire coding region.

[0079] The plasmid was transferred into BL21(DE3) competent E. coli cells. A positive colony can be inoculated into 1 L LB medium and incubated at 37°C with 200 rpm rotation until an OD600 of 0.6 was reached. Then, 1 mM of inducer IPTG can be added to induce expression at 16°C for 18 hours. Cells can be harvested and suspended in lysis buffer (25 mM Tris-HCl pH=7.5, 0.15 M NaCl, 20 mM imidazole, 2 mM β-mercaptoethanol, and 1× Roche proteinase inhibitor cocktail). Cells can be lysed by sonication, and debris can be spun down at 50,000 g for 1 hour.

[0080] Proteins can be isolated by first passing the lysate through a Ni column. The column can be washed with 50 column volumes (CV) of binding buffer (lysis buffer without proteinase inhibitors) and then 10 CV of wash buffer (binding buffer with a total of 40 mM imidazole). Proteins can be eluted with elution buffer (binding buffer with a total of 300 mM imidazole).

[0081] The crude extract can then be further purified by size exclusion chromatography using a Superdex 200 incremental 10 / 300 column containing 25 mM Tris-HCl pH 7.5, 0.15 M NaCl, 5% glycerol, and 1 mM DTT. The final product was stored in 50-100 μl aliquots at -80°C.

[0082] Figure 1 shows the time course of the proportional abundance of relevant gut microbiome species in healthy volunteers or patients with CDI receiving a DNA pol IIIC inhibitor. Using primers targeting Clostridium cluster XIVa (C. coccoides) and Clostridium cluster IV (C. leptum), qPCR was used to quantify the proportion of these related Firmicutes over time. As seen in Figure 1A, samples collected from a 12-day phase 1 study of healthy volunteers showed that twice-daily treatment with 450 mg of a small-molecule DNA pol IIIC inhibitor increased the abundance of C. coccoides. Clostridium cluster XIVa, also known as the Clostridium coccoides group, consists of 21 species. These commensal bacteria may play an important role in gut homeostasis. Furthermore, members of the XIVa cluster have been shown to attenuate inflammation and allergic diseases, and the cellular components and metabolites of these species, such as butyrate, secondary bile acids, and indolepropionic acid, may play a probiotic role in the gut, primarily by activating intestinal epithelial cells, strengthening the intestinal barrier, and interacting with the immune system.

[0083] As shown in Figure 1B, participants with CDI constituted the cohort in Phase 2a of this study. In contrast to healthy volunteers in Phase 1 of the trial, Phase 2a subjects had lower abundances of both Clostridium cluster XIVa and cluster IV throughout the study. However, Tables 2 and 3 show that Actinobacteria increased in abundance after initiation of DNA pol IIIC inhibitors (primarily Bifidobacteriales or Coriobacteriales) and persisted throughout the entire treatment period. Compared to the Phase 1 study, the baseline microbiota in the Phase 2a CDI study had lower proportions of Actinobacteria and Firmicutes, and increased Bacteroidetes. In CDI patients, Actinobacteria increased in abundance (primarily Coriobacteriales), followed within 2–3 days by a decrease in Bacteroidetes abundance and an increase in Lachnospiraceae and Ruminococcaceae abundance. Furthermore, both Phase 1 and Phase 2a studies showed that the proportion of Lachnospiraceae was conserved, as was the abundance of Clostridiales.

[0084] Broth microdilution-based determination of minimum inhibitory concentrations (MICs) On the day of testing, compounds may be dissolved in pure DMSO (Sigma 276855-2L) to a stock concentration of 20 mM. In a v-bottom 96-well plate (Axygen-wipp02280), 30 μl of DMSO was added to wells 1 through 12 by manual pipetting. To well 1, 30 μl of compound DMSO stock (20 mM) may be added and mixed by pipetting. Two-fold serial dilutions may be performed by transferring and mixing 30 μl of solution from well 1 to well 2, then from well 2 to well 3, etc., up to well 11. Well 12 may be filled with 30 μl of DMSO without compound. This may be the "mother plate" for the drug. From well 1 to well 12, the drug concentrations in the mother plate can be 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.156, 0.078, 0.039, 0.02, 0.01, and 0 mM in DMSO. A multi-pipette can be used to perform serial dilutions. Concentrations can be adjusted according to compound potency. The Echo® Acoustic Liquid Handling System can be used to create daughter plates, replacing manual pipetting.

[0085] One day before MIC testing, bacterial strains may be streaked onto MHA plates from -80°C glycerol stocks and incubated at 37°C for 20 hours. Streptococcus pneumoniae may be streaked onto blood agar and incubated at 37°C, 5% CO2. A single colony was picked using an inoculation loop (Greiner-731175) and suspended in 5 ml of sterile saline. The turbidity of the suspension was adjusted to approximately 1.0 x 10 8 The bacterial suspension may be diluted 100-fold with the corresponding test medium (Table 1), which can be used to inoculate daughter plates.

[0086] To prepare U-bottom 96-well "daughter plates" (Costar 3788), 98 μl of test medium was added to each well of the daughter plate. Aliquots of 2 μl of solution from the mother plate may then be transferred in duplicate to the daughter plates using a multi-pipette.

[0087] Aliquots of 100 μl of the bacterial suspension may be inoculated into each well of the daughter plate using a multi-pipette. Each well contains approximately 5.0 × 10 bacteria in 200 μl of the corresponding test medium. 5 cfu / ml of bacteria, 1% DMSO, and serially diluted compounds at 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.2, 0.1, and 0 μM from well 1 to well 12, respectively.

[0088] The plates may be incubated in ambient atmosphere and in a 37° C. incubator for 20 hours.

[0089] MIC values ​​were determined by visual inspection as the lowest compound concentration that completely or significantly inhibited bacterial growth in the test medium.

[0090] The compounds of the present invention were tested for antibacterial activity against various bacterial organisms, including Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Streptococcus pneumoniae, and Escherichia coli. The compounds described in Examples 1, 18, 19, 28, 30, 32, and 34 had K values ​​of 0.31-1.45 μM against the Bacillus subtilis DNA pol IIIC enzyme and MIC values ​​of 0.25-4.0 μg / ml against strains of Gram-positive organisms. The compound exhibited weak Gram-negative activity against Escherichia coli with MICs of 16->64 μg / ml.

[0091] Figure 2 shows the results of MIC measurements performed on selected Firmicutes isolated from samples collected during Phase 2a of the study. Susceptibility to DNA pol IIIC inhibitors was found to vary among the beneficial Firmicutes strains tested. Surprisingly, susceptibility not only varied among heterologous groups of different species but also among isolates belonging to the same species (Table 4). Isolated Clostridium butyricum strains (C. butyricum 1008 and C. butyricum 1007) were sequenced and compared. As seen in Figure 2A, C. butyricum 1008 (1.5 μg / mL) exhibits much lower susceptibility than C. butyricum 1007. In contrast to C. butyricum 1008, C. butyricum 1007 showed attenuated susceptibility (C. butyricum 1007 >100 μg / mL). Figure 2B is a rendering of the molecular structure of the two C. butyricum strains. As seen in Table 1 below, compared to the Clostridium butyricum reference strain, one SNP was identified in the susceptible strain C. butyricum 1008, Y240D (Tyr240Asp), and two SNPs were identified in the less susceptible strain C. butyricum 1007, Y38D and D146E (Tyr38Asp and Asp146Glu). These results suggest that small molecule inhibitors of DNA pol IIIC offer enhanced selectivity against low-G+C Gram-positive pathogenic symbionts. Furthermore, inhibitors such as the provided LDN analogs may circumvent antibiotic resistance against targeted pathogenic symbionts.

[0092] [Table 1]

[0093] Furthermore, results suggest that specific beneficial Firmicutes or commensals exhibited varying sensitivities to small-molecule inhibitors of DNA pol IIIC. Using isolated gut microbiota species, DNA pol IIIC inhibitors were inactive (MIC > 64 μg / mL) against representative Actinobacteria (Bifidobacteriaceae and Coriobacteriaceae) and certain Firmicutes (Lachnospiraceae and Lactobacillaceae), but were highly active (MIC ≤ 2 μg / mL) against strains of C. difficile.

[0094] Example 2: Microbiome Research for Phase I Healthy Volunteer Study: BACKGROUND: A healthy gut microbiome is composed of two major bacterial groups, called phyla. Firmicutes (Gram-positive spore-forming organisms) and Bacteroidetes (Gram-negative non-spore-forming organisms) are the most common. A third phylum, Proteobacteria (Gram-negative facultative anaerobes), is present at low abundance but typically constitutes 2-5% of a healthy microbiome. A fourth phylum, Actinobacteria (mostly Gram-positive, mostly saprophytic bacteria), is present in large proportions in children and may generally decrease in overall proportion with age. Patients suffering from C. difficile infection are in a state of dysbiosis, and such patients often have an increased proportion of Proteobacteria, e.g., an excess of Proteobacteria or "Proteobacteria dysbacteria," and decreased numbers of Firmicutes and Bacteroidetes.

[0095] Ibezapolstat Study: Using stool samples obtained from a Phase 1 healthy volunteer study and shotgun metagenomic sequencing, it was demonstrated that treatment with ivezapolstat for 10 days resulted in a significantly different microbiome profile in subjects compared to subjects receiving placebo. The difference was an increased proportion of Proteobacteria in vancomycin-treated subjects, versus an increased proportion of Actinobacteria and Firmicutes in ivezapolstat-treated subjects.

[0096] Methods and Materials Clinical Trial Description: Six healthy volunteers received 450 mg of ibezapolstat twice daily for 10 consecutive days. Stool samples were collected daily (Garey KW, et al. A randomized, double-blind, placebo-controlled, single- and multiple-ascending-dose Phase 1 study to determine the safety, pharmacokinetics, and food and faecal microbiome effects of ibezapolstat administered orally to healthy subjects. J Antimicrob Chemother 2020;75(12):3635-3643). Institutional review board approval was obtained (Midlands Institutional Review Board IRB #222220170383), and all volunteers signed informed consent forms before any study procedures were performed. For this analysis, subjects receiving 450 mg of ibezapolstat twice daily had daily stool samples collected from baseline through day 0, day 13, and day 30 of follow-up, if available. Stool samples were immediately frozen at -80°C and then shipped on dry ice to the University of Houston for analysis.

[0097] Stool DNA extraction and shotgun metagenomic sequencing: Stool DNA was extracted using the DNAeasy Power Soil Pro kit (Qiagen, catalog number 1288-100) on a QiaCube automated DNA extraction system as previously described (Garey KW, et al. A randomized, double-blind, placebo-controlled, single, and multiple ascending dose Phase 1 study to determine the safety, pharmacokinetics, and food and faecal microbiome effects of ibezapolstat administered orally to healthy subjects. J Antimicrob Chemother 2020;75(12):3635-3643). Shotgun metagenomic sequencing was performed at the University of Houston Sequencing and Gene Editing Core (Houston, TX, USA) using the Nextera DNA Flex Library Prep Kit for DNA library preparation and the Illumina NextSeq 500 platform for sequencing. CLC Genomic Workbench version 12 (Qiagen) was used for metagenomic assembly and generation of abundance tables.

[0098] 16S ribosomal RNA (rRNA) gene sequencing: Microbial taxonomy was characterized using 16S rRNA sequencing as described in Gonzales-Luna (2021). The V3-V4 region of the 16S rRNA gene was sequenced using an Illumina-based sequencing platform with a minimum of 15,000 reads per sample to assess gut microbiome community structure. Quality-filtered sequence reads with at least 97% similarity were clustered into Operational Taxonomic Units (OTUs), and representative sequences from each OTU were assigned taxonomic identity at the species level by searching against the NCBI 16S rRNA sequence database (published September 1, 2018) using the NCBI BLAST+ package v2.8.1 2018.

[0099] Shotgun metagenomic sequencing: We performed shotgun metagenomic sequencing on DNA extracted from fecal samples previously used for 16S rRNA sequencing using an Illumina-based platform for the analysis of microbiome functional genes. Functional gene profiling of the shotgun metagenomics was performed using the HUMAnN2 v0.11.2 pipeline. The preprocessing step involved quality filtering of the sequencing reads, followed by screening and removal of contaminating host (human) reads. Trimmomatic v0.38 was used to filter and trim the raw sequence data with default cutoff settings. Reads were searched against the human genome database in paired-end mode using the bowtie2 algorithm and discarded if they mapped to the database. To obtain gene family profiles, these quality-controlled metagenomic sequences were first searched against a nucleotide database (ChocoPhlAn) using bowtie2, and then against a protein database (UniRef90) using diamond. All identified gene families were annotated using UniRef90, and pathways were annotated using MetaCyc identifiers.

[0100] Microbiome analysis Using the sequencing described above, we analyzed the time-dependent changes in the proportional abundance of gut microbiome species in healthy volunteers receiving ibezamis. Metagenomic data showing the proportion of Firmicutes isolated per day during the study are shown in Table 2 below. The baseline gut microbiota from healthy volunteers was primarily Firmicutes, Bacteroidetes, or Actinobacteria. Actinobacteria increased in abundance after IBZ initiation (primarily Bifidobacteriales or Coriobacteriales) and persisted throughout the treatment period. As shown in Table 2 below, the most commonly isolated genera (species) during the Phase 1 study of healthy volunteers were Streptococcusaceae (Lactococcus lactis and Streptococcus thermophilus), Lachnospiraceae (Blautia, Roseburia), and Ruminococcaceae (Faecalibacterium, Ruminococcus).

[0101] [Table 2]

[0102] Example 3: Microbiome data from a Phase 2a clinical trial of ivezapolstat for CDI Phase 2 clinical trials: A phase 2 clinical trial was designed to evaluate ibezapolstat in the treatment of CDI.

[0103] Phase 2a of this clinical trial was an open-label cohort of 10 subjects from a US research center. In this cohort, 10 patients with mild or moderate C. difficile-induced diarrhea, diagnosed via toxin EIA+, were treated with ibezapolstat 450 mg orally twice daily for 10 days. All patients were followed for recurrence over a 28±2-day period. Stool samples were collected throughout the course of treatment and at follow-up. Patients' stool samples were evaluated for C. difficile culture and microbiome changes. The study demonstrated 100% clinical cure at day 12 and 100% sustained clinical cure at day 38. Favorable microbiome changes included overgrowth of species from the phyla Actinobacteria and Firmicutes during treatment. These findings support that the effects of the microbiome may portend favorable patient outcomes, including low recurrence rates: 100% clearance of infection, no recurrence of infection (100%), and an acceptable adverse event profile.

[0104] Methods and Materials Safety evaluation Safety assessments included AE assessment, physical examination, vital signs, clinical laboratory tests (chemistry, hematology, and urinalysis), and electrocardiography. Safety endpoints, including the nature, frequency, and severity of AEs, were recorded for all subjects. AEs were assessed at each visit beginning at enrollment and classified according to the Medical Dictionary for Regulatory Activities (MedDRA version 15.0). AE severity (mild, moderate, or severe) and causality (unrelated, possibly related, or probably related to the study drug) were assessed by investigators at each site.

[0105] microbiology Stool samples were cultured for C. difficile growth on selective cycloserine-cefoxitin fructose agar (CCFA) at 37°C under anaerobic conditions for 48 hours. (Gonzales-Luna AJ, Carlson TJ, Dotson KM, et al. PCR ribotypes of Clostridioides difficile across Texas from 2011 to 2018, including emergence of ribotype 255. Emerg Microbes Infect 2020;9(1):341-7) Isolates were identified as C. difficile based on growth and morphology and confirmed by PCR for C. difficile toxins and tpi genes. C. difficile was strain-typed using a PCR-based ribotyping method as previously described (Gonzales-Luna AJ, Carlson TJ, Dotson KM, et al. PCR ribotypes of Clostridioides difficile across Texas from 2011 to 2018 including emergence of ribotype 255. Emerg Microbes Infect 2020;9(1):341-7.). The minimum inhibitory concentration (MIC) for ibezazolstat was determined by broth microdilution in 0.1% sodium taurocholate Brain Heart Infusion (BHI) medium (Begum K, Basseres E, Miranda J, et al. In Vitro Activity of Omadacycline, a New Tetracycline Analog, and Comparators against Clostridioides difficile. Antimicrob Agents Chemother. 2020;64(8)).

[0106] statistical analysis An intent-to-treat analysis was performed for patients receiving at least one dose of ivezapolstat. Descriptive statistics were calculated for efficacy, safety / tolerability, and PK data generated using SAS version 9.4 software (SAS Institute, Inc., Cary, NC, USA). Microbiome summary plots and data visualizations were created using R software version 4.1.1 (R Core Team 2021, Vienna, Austria). (R Core Team (2013). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria). Proportional changes in bacterial taxa over the 10-day dosing interval were calculated using a linear regression model for taxa with a proportional change of at least 1 percent over the study period. A p-value of <0.05 was considered significant.

[0107] Phase 2a Clinical Trial Results: A completed Phase 2a CDI clinical trial demonstrated a 100% success rate in favorable microbiome changes. Phase 2a data demonstrated complete eradication of colonic C. difficile by day 3 of treatment with ivezapolstat, as well as an overgrowth of healthy gut microbiota, Actinobacteria species, and Firmicutes species during and after treatment. Additionally, data demonstrated an increase in the proportion of healthy microbiota, including Clostridiales taxa. The baseline microbiota in the Phase 2a CDI study had a low proportion of Actinobacteria and Firmicutes, with an increase in Bacteroidetes. In CDI patients, Actinobacteria increased in abundance after the initiation of IBZ (mainly Coriobacteriales), followed within 2–3 days by a decrease in the abundance of Bacteroidetes and an increase in the abundance of Lachnospiraceae and Ruminococcaceae.

[0108] patient Ten patients aged 27 to 75 (±15) years (50% female; 100% Caucasian; 80% Hispanic or Latino ethnicity) were enrolled. All 10 patients received ibezamisamide, and all 10 patients completed the study. The median number of unformed stools within 24 hours before starting treatment was 4 (range: 3 to 10). Two of the 10 patients had received antibiotic treatment with either metronidazole or vancomycin for less than 24 hours before starting ibezamisamide. No patients were hospitalized before or after enrollment.

[0109] Microbiological results Metagenomic data from the Phase 2a study showing the proportion of Firmicutes isolated per day during the study are shown in Table 3. In CDI patients, Actinobacteria increased in abundance after the initiation of IBZ (mainly Coriobacteriales), followed within 2–3 days by a decrease in the abundance of Bacteroidetes and an increase in the abundance of Lachnospiraceae and Ruminococcaceae. The genera (species) most commonly isolated during the study included the family Streptococcaceae (Streptococcus salivarus and Streptococcus thermophilus), the family Lachnospiraceae (Blautia, Roseburia), and the family Faecalibacterium (Ruminococcaceae). [Table 3]

[0110] Example 4: Gram-positive selectivity spectrum of ivezapolstat The purpose of this study was to evaluate the selectivity of ibezazolstat against Gram-positive gut microbiota. In vitro and human studies demonstrated potent activity of ibezazolstat against C. difficile, while selective activity against other beneficial Gram-positive gut microbiota may reduce the risk of recurrent CDI.

[0111] Methods and Materials Firmicutes fecal isolate Using stool samples and microbiome data from the Phase 1 and Phase 2a studies outlined above, changes in the proportional abundance of gut microbiome species were analyzed over time in healthy volunteers and patients with CDI receiving ibezapolstat. In Phase 1, six healthy volunteers received 450 mg of ibezapolstat twice daily for 10 days. For Phase 1, subjects receiving 450 mg of ibezapolstat twice daily collected daily stool samples, if available, from baseline to day 13 and for follow-up periods of day 30. Stool samples were immediately frozen at -80°C and then shipped on dry ice to the University of Houston for analysis.

[0112] Phase 2a of this trial was an open-label cohort of 10 subjects from a US research center. In this cohort, 10 patients with mild or moderate C. difficile-induced diarrhea, diagnosed via toxin EIA+, were treated with ibezapolstat 450 mg orally twice daily for 10 days. Stool samples were collected during the course of treatment and at follow-up.

[0113] Stool DNA extraction and shotgun metagenomic sequencing: Stool DNA was extracted using the DNAeasy Power Soil Pro kit (Qiagen, catalog number 1288-100) on a QiaCube automated DNA extraction system as previously described (Garey KW, et al. A randomized, double-blind, placebo-controlled, single- and multiple-ascending-dose Phase 1 study to determine the safety, pharmacokinetics, and food and faecal microbiome effects of ibezapolstat administered orally to healthy subjects. J Antimicrob Chemother 2020;75(12):3635-3643). Shotgun metagenomic sequencing was performed at the University of Houston Sequencing and Gene Editing Core (Houston, TX, USA) using the Nextera DNA Flex Library Prep Kit for DNA library preparation and the Illumina NextSeq 500 platform for sequencing. CLC Genomic Workbench version 12 (Qiagen) was used for metagenomic assembly and generation of abundance tables.

[0114] 16S ribosomal RNA (rRNA) gene sequencing: Microbial taxonomy was characterized using 16S rRNA sequencing as described in Gonzales-Luna (2021). The V3-V4 region of the 16S rRNA gene was sequenced using an Illumina-based sequencing platform with a minimum of 15,000 reads per sample to assess gut microbiome community structure. Quality-filtered sequence reads with at least 97% similarity were clustered into Operational Taxonomic Units (OTUs), and representative sequences from each OTU were assigned taxonomic identity at the species level by searching against the NCBI 16S rRNA sequence database (published September 1, 2018) using the NCBI BLAST+ package v2.8.1 2018.

[0115] Shotgun metagenomic sequencing: We performed shotgun metagenomic sequencing on DNA extracted from fecal samples previously used for 16S rRNA sequencing using an Illumina-based platform for the analysis of microbiome functional genes. Functional gene profiling of the shotgun metagenomics was performed using the HUMAnN2 v0.11.2 pipeline. The preprocessing step involved quality filtering of the sequencing reads, followed by screening and removal of contaminating host (human) reads. Trimmomatic v0.38 was used to filter and trim the raw sequence data with default cutoff settings. Reads were searched against the human genome database in paired-end mode using the bowtie2 algorithm and discarded if they mapped to the database. To obtain gene family profiles, these quality-controlled metagenomic sequences were first searched against a nucleotide database (ChocoPhlAn) using bowtie2, and then against a protein database (UniRef90) using diamond. All identified gene families were annotated using UniRef90, and pathways were annotated using MetaCyc identifiers.

[0116] Quantitative PCR (qPCR) analysis The quantity and quality of extracted DNA were assayed using a Qubit 4 Fluorometer (Invitrogen). Sample DNA was diluted to 5ng / μL with PCR-grade water. DNA levels of bacterial groups were assessed using specific PCR primers / conditions. 11~14qPCR was performed on each sample in triplicate in a final volume of 20 μL containing 25 ng of DNA template, 0.5 μM primers, and QuantiTect SYBR Green Mixes (Qiagen) using a 7300 Real-Time PCR System (Applied Biosystems). For eubacteria, 0.25 μM FAM-tagged probe and TaqPath ProAmp Master Mixes (Qiagen) were used. Threshold cycle values ​​were converted to copy numbers per ng of DNA using a standard curve. Standards were prepared by PCR using species-specific primers on DNA from the appropriate bacterial strain or normal stool. PCR products were cloned using the Invitrogen TOPO PCR Cloning Kit (Invitrogen) and verified by sequencing at the University of Houston Core Facility. Basic Local Alignment Search Tool (BLAST) searches were performed to identify the closest matching database sequences. Ten-fold serially diluted plasmid standard DNA (5 × 10 8 A range of 1000 copies (~500 copies) was run in triplicate on each qPCR plate. 2 Values ​​were calculated relative to the standard. Copies per gram of stool were calculated taking into account the initial sample DNA concentration and stool weight. The change in bacterial levels (Δlog10 copies / gram stool) from the entrance level to each available successive time point was determined for each participant, and the median change was calculated.

[0117] Firmicutes Isolates (Phase 2a Samples): Fecal samples were homogenized in reduced PBS (0.1 g feces / ml PBS), serially diluted, and directly plated onto YCFA7 agar supplemented with 0.002 g / ml glucose, maltose, and cellobiose, respectively, in large (13.5 cm diameter) Petri dishes. Isolated colonies were identified by PCR amplification of the full-length 16S rRNA gene (using the 7F (5'-AGAGTTTGATYMTGGCTCAG-3') forward primer and the 1510R (5'-ACGGYTACCTTGTTACGACTT-3') reverse primer) followed by Sanger sequencing. Full-length 16S rRNA gene sequence reads were aligned to Operational Taxonomic Units (OTUs) using the Ribosomal Database Project (RDP) at CLC Genomics (Qiagen). Full-length 16S rRNA gene sequences of OTUs at various levels were compared with the Ribosomal Database Project (RDP) reference database to assign taxonomic names to the genus level, and BLAST searches were performed to identify any candidate novel species.

[0118] Whole genome sequencing Genomic DNA was extracted from at least one representative of each identified species. DNA was sequenced on the Illumina HiSeq platform, generating 150-bp reads that were assembled and annotated for further analysis. For whole-genome SNP analysis, cleaned sequence reads were mapped to the reference genome using CLC Genomics and the RedDog pipeline according to the developer's guidelines (https: / / github.com / katholt / RedDog). Briefly, Bowtie2 version 2.2.3 was used for mapping, and SAMtools version 0.1.19 was used for SNP calling. Only high-quality SNPs were used for phylogenetic analysis.

[0119] Determination of minimum inhibitory concentration (MIC): Strains were plated onto blood agar (Hardy Diagnostics) for 24 hours of incubation at 37°C in an anaerobic environment (Coy vinyl anaerobic chamber). After incubation, 3-5 well-isolated colonies were suspended in 5 mL of BHI broth (Criterion Media). The cultures were incubated in the anaerobic chamber for 24 hours to achieve a 0.5 McFarland standard. Brucella agar containing 5% hemin (5 μg / mL) (Sigma), vitamin K (10 μg / mL), and defibrinated sheep blood (Northeast Lab Services) was then prepared. The supplemented Brucella agar was then used in two-fold serial dilutions to produce plates with IBZ concentrations ranging from 0.5 to 16 μg / mL. The plates were then allowed to dry in a sterile area. After drying, the plates were covered with foil to prevent light damage to the hemin and placed in an anaerobic chamber for 1 hour to reduce the medium. The broth cultures were then spotted onto plates and covered with foil. After the plates were allowed to dry completely, they were inverted for a 48-hour incubation period. The plates were analyzed and the MICs recorded.

[0120] Microbiology results: The baseline gut microbiota from healthy volunteers was predominantly Firmicutes, Bacteroidetes, or Actinobacteria. Actinobacteria increased in abundance (primarily Bifidobacteriales or Coriobacteriales) after initiation of ivezapolstat and persisted throughout the treatment period. The proportion of Lachnospiraceae and the abundance of Clostridiales were preserved in both the Phase 1 and Phase 2a studies. Compared with the Phase 1 study, the baseline microbiota in the Phase 2a CDI study had lower proportions of Actinobacteria and Firmicutes and increased Bacteroidetes. In CDI patients, Actinobacteria increased in abundance after initiation of ivezapolstat (mainly Coriobacteriales), followed within 2–3 days by a decrease in the abundance of Bacteroidetes and an increase in the abundance of Lachnospiraceae and Ruminococcaceae.

[0121] Beneficial Firmicutes exhibit mixed susceptibility to ibezapolstat Using isolated gut microbiota species, ibezazolstat was inactive (MIC >64 μg / mL) against representative Actinobacteria (Bifidobacteriaceae and Coriobacteriaceae) and certain Firmicutes (Lachnospiraceae and Lactobacillaceae), but was highly active against C. difficile strains (MIC <2 μg / mL). Therefore, microbiome changes induced by ibezazolstat may depend on the composition of the underlying baseline microbiome. However, in both Phase 1 and Phase 2a cohorts, the abundance of Actinobacteria increased after treatment initiation. The IBZ microbiome data, combined with in vitro MIC measurements, demonstrated the persistence or repopulation of a healthy microbiota associated with beneficial physiological effects. The results of MIC determinations for Firmicutes are shown in Table 4. Among the beneficial Firmicutes isolated, ibexapolstat showed mixed susceptibility.

[0122] [Table 4]

[0123] Although Firmicutes generally possess DNA Pol IIIC enzymes, some members of the Firmicutes phylum (also known as Bacillus) were more susceptible to ibezazolstat. Among the heterogeneous population of Firmicutes tested, different strains of Clostridium butyricum exhibited differential susceptibility. As seen in Figure 1A, C. butyricum 1008 was more susceptible to ibezazolstat (IBZ MIC: 1.5 μg / mL) compared to C. butyricum 1007 (IBZ MIC: >100 μg / mL). Following whole-genome sequencing, three single nucleotide polymorphisms (SNPs) were identified. As seen in Table 1, compared to the reference strain of Clostridium butyricum, one SNP was identified in the susceptible strain C. butyricum 1008, Y240D (Tyr240Asp), and two SNPs were identified in the less susceptible strain C. butyricum 1007, Y38D and D146E (Tyr38Asp and Asp146Glu). The results suggest that targeted ivezapolstat drug development against C. difficile may result in intermittent activity against beneficial commensals.

[0124] Example 5: Novel Pharmacology and Susceptibility of Ibezapolstat against C. difficile Isolates with Reduced Susceptibility to C. difficile-Directed Antibiotics The objective of this study was to evaluate the efficacy of ibezazolstat against C. difficile strains that exhibit reduced susceptibility to current CDI antibiotics. Additionally, this study evaluated the effect of IBZ treatment on motility inhibition and flagellar gene expression in C. difficile strains with reduced susceptibility to current CDI antibiotics.

[0125] Methods and Materials The objectives of this study were to evaluate the susceptibility of IBZ to strains with reduced susceptibility to current CDI antibiotics and to assess motility inhibition.

[0126] Isolate: Twelve clinical isolates with reduced susceptibility to metronidazole (MIC range: 0.25-8 ug / mL), vancomycin (MIC range: 1-16 ug / mL), or fidaxomicin (<0.03125-2 ug / mL) were tested.

[0127] Determination of minimum inhibitory concentration (MIC): Agar dilution MIC studies were performed on C. difficile strains with reduced susceptibility to metronidazole, vancomycin, and fidaxomicin according to the guidelines in CLSI document M11-A7 for anaerobic bacteria. C. difficile cultures were prepared by inoculating a single colony of bacteria into BHI supplemented with 0.1% sodium taurocholate. After 24 hours of incubation at 37°C in an anaerobic chamber, the preculture was diluted 1:100 to approximately 10% in fresh BHI containing the appropriate antibiotic concentration. 6 The antibiotics were diluted to CFU / mL. Antibiotic concentrations were prepared by a two-fold dilution series (one concentration per agar plate) according to CLSI guidelines. For example, for each of the three antibiotics, namely vancomycin, ibexapolstat, and metronidazole, the antibiotic concentrations ranged from approximately 64 μg / mL to approximately 0.25 μg / mL, and for fidaxomicin, the concentrations ranged from approximately 16 μg / mL to approximately 0.03 μg / mL.

[0128] Inhibitory concentrations were determined by ocular visualization at 24 hours. As shown in Table 5, MIC values ​​are related to the reduced susceptibility of the antibiotic (difference >8 × MIC value), and MICs are expressed in mg / L. The results demonstrate that IBZ maintains its efficacy against clinical isolates with reduced susceptibility to metronidazole, vancomycin, and fidaxomicin. Furthermore, IBZ MIC50 and MIC90 did not differ between susceptible and reduced susceptible isolates. [Table 5]

[0129] Quantitative PCR (qPCR) analysis C. difficile motility was assessed using quantitative RT-PCR. Following pretreatment with sub-MIC concentrations of IBZ, adapted from the methodology of Doan et al. (Antibiotics 2022), relevant flagellar gene expression (fliA, flgB, fliC-VIP) of the reference C. difficile strain CD630 was quantified by qPCR. Briefly, bacterial precultures were diluted and then grown for 4 hours in BHI at a sub-inhibitory concentration of IBZ (0.5× the MIC) under anaerobic conditions at 37°C. Transcription levels of fliA, flgB, and fliC were determined and compared to the control gluD recombinant protein (Clostridium difficile).

[0130] Figure 3 shows the target genes on the x-axis and the relative expression of these genes compared to the untreated control on the y-axis. Furthermore, to better visualize the data, the relative expression of the control was normalized to a value of 1. Thus, Figure 3 shows that, compared to the untreated control, when treated with IBZ, fliA expression was reduced by approximately 60%, flgB expression was reduced by approximately 30%, and fliC expression was reduced by approximately 80%. Thus, qPCR analysis revealed an overall 2-5-fold decrease in flagellar gene expression after sub-MIC IBZ exposure.

[0131] Motility assay: C. difficile motility was assessed using a phenotypic motility assay. Briefly, cultures of CD 630 were prepared by inoculating BHI medium containing 0.3% agar and a subinhibitory concentration of IBZ and grown anaerobically at 37°C for 48 hours. After incubation, bacterial strains were plated on semi-solid BHI agar for 4 hours and visually observed for movement.

[0132] Figure 4 shows the results of the motility assay. As can be seen in Figure 4, the control strain is able to swim without constraint and therefore moves freely away from the inoculation point. In contrast, strains exposed to subinhibitory (non-lethal) concentrations of IBZ in the medium remain near their inoculation point. These results indicate that CD 630 cultures are less motile due to the presence of IBZ in the medium.

[0133] The results showed that IBZ maintained activity against C. difficile strains and exhibited reduced susceptibility to other commonly used antibiotics, demonstrating novel pharmacological properties likely attributable to its unique mechanism of action.

[0134] Example 6: Metagenomic Evaluation of Ibezapolstat Compared to Other Anti-Clostridioides difficile Agents Published studies have demonstrated that treatment with ibezazolstat (IBZ) can result in less microbiome disruption than treatment with vancomycin in humans (see, for example, Figure 5). Despite this, there are no comparative microbiome studies on other anti-C. difficile antibiotics. The purpose of this study was to compare in vivo changes in the gut microbiome in response to treatment with IBZ and other anti-C. difficile antibiotics. Therefore, this study set out to compare gut microbiome disruption from IBZ with three other anti-C. difficile antibiotics, including vancomycin (VAN), fidaxomicin (FDX), or metronidazole (MTZ).

[0135] Methods and Materials Germ-free (GF) mice (6 per group) were randomly assigned to IBZ, VAN, FDX, MTZ, or control groups. Figure 6 shows the basic experimental design of the humanized germ-free mouse study.

[0136] Germ-free (GF) humanized mice GF mice were fed a fecal slurry from healthy humans via oral gavage. The humanized mice were housed in a sterile biological safety cabinet (BSC) for one week to allow the donor gut microbiome to establish. The humanized mice were then transferred to sterile microisolator cages, and the pelleted chow diet was replaced with a powdered chow diet. The humanized mice were then allowed to acclimate to the powdered chow diet for one week. Additionally, a stool sample was obtained before the diet change (Baseline 1). On Day 14, once the mice had acclimatized to the powdered chow diet, a second baseline stool sample was obtained (Baseline 2), and antibiotic treatment was initiated. The appropriate antibiotic was added to the powdered chow diet for 10 days. On Day 16, two days after antibiotic treatment began, another stool sample was collected (ABX-1). During the 10-day antibiotic treatment, the powdered chow dish was replenished daily with fresh chow containing the randomly assigned antibiotic. Stool samples were collected again on Day 24 (ABX-2).

[0137] Microbiome analysis To assess the microbiome effects of antibiotics, 16S rRNA metagenomics was used on stool samples collected on day 0 (baseline 1 and baseline 2) and 2 and 14 days after antibiotic treatment. Bulk DNA was extracted from stool samples using the DNeasy® PowerSoil® kit. The V4 region of the bacterial 16S rRNA gene was sequenced using Illumina MiSeq and CLC Genomics Workbench. Raw sequencing reads were processed and curated using the mothur (v. 1.48.0) software package, and the mothur MiSeq SOP was performed as outlined in Schloss, PD, et al., "Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities." Appl Environ Microbiol, 2009, 75(23):7537-41 (see also Kozich, JJ, et al., "Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform." Appl Environ Microbiol, 2013, 79(17):5112-20). All data visualization and statistical analysis were performed using R studio (R Core Team (2023). R: A Language and Environment for Statistical Computing, Vienna, Austria).

[0138] result Before antibiotic initiation, Shannon index alpha diversity was similar between treatment groups.

[0139] Microbiome analysis Figures 7A–E show boxplots with lines connecting groups based on color, where the color represents the antibiotic to which the mice were exposed during the last 10 days of the experiment (or none in the case of the no-drug control (ND control) and baseline samples). Changes in gut microbiome alpha diversity throughout the experiment are shown in Figures 7A and 7B, which show the change in abundance of Operational Taxonomic Units (OTUs) within the gut microbiome over the course of the experiment (Figure 7A), as well as the change in inverse Simpson's index within the gut microbiome over the course of the experiment (Figure 7B). For both metrics, the combination of dietary modification followed by exposure to different antibiotics significantly affected the inverse Simpson's index (ANOVA; p<0.001). As seen in Figures 7A and 7B, there was a significant decrease in alpha diversity in all antibiotic groups compared to the control (p<0.05).

[0140] Figures 7C, 7D, and 7E demonstrate changes in gut microbiome beta diversity (group diversity) throughout the experiment, showing clear clustering in IBZ-fed mice relative to all other antibiotics. Figure 7C shows the change in gut microbiome beta dispersion (centroid distance) throughout the experiment, Figure 7D shows the change in Bray-Curtis dissimilarity relative to baseline 1 (D7) throughout the experiment, and Figure 7E shows the change in Bray-Curtis dissimilarity relative to baseline 2 (D14). For all metrics of beta diversity, the diet change and subsequent antibiotic exposure significantly increased Bray-Curtis dissimilarity relative to baseline 2 (D14) (ANOVA, all p<0.001).

[0141] Figure 7F provides the non-metric multidimensional scaling (NMDS) of the Bray-Curtis dissimilarity.

[0142] The boxplots shown in Figures 7A-7E include dots (shown at the top of each plot) representing one sample collected at each time point; the size indicates the inverse Simpson's index value for that sample, the shape indicates which trial the sample was collected from, and the color indicates which antibiotic (or lack thereof) each sample was exposed to. When comparing the exposure groups (color) at the two time points where there was an antibiotic-exposed group (ABX-1 and ABX-2), there was a statistical difference between the antibiotic-exposed groups (PERMANOVA; p<0.001 for both time points). Furthermore, when comparing Trial 1 and Trial 2, there was a statistical difference (PERMANOVA; p<0.001) between the two trials at all time points (Baseline 1, Baseline 2, ABX-1, and ABX-2). Table 6 below shows the changes in gut microbiome diversity metrics observed in treated humanized mice, demonstrating the comparisons made between antibiotic treatments. For each antibiotic (ibezapolstat, fidaxomicin, vancomycin, and metronidazole), comparisons were made with baseline 2 (D14). Comparisons between D7 and D14 to account for changes in diversity due to dietary changes were made using baseline 1 (D7) as the reference point. Significance symbols were determined based on the following p-value parameters: approximately 0 = "***", approximately 0.001 = "**", approximately 0.01 = "*", approximately 0.05 = "." Any p-values ​​> 0.05 were not considered significant.

[0143] [Table 6]

[0144] Figures 8A-8D provide stacked bar graphs showing the average relative abundance (expressed as percentages) of different bacterial taxonomic levels across the entire experiment. Thus, Figure 8A is at the phylum level, Figure 8B is at the class level, Figure 8C is at the order level, and Figure 8D is at the family level. Furthermore, Table 7 below shows the changes in relative abundance of bacterial phyla within the gut microbiome observed in treated humanized mice and a comparison between different antibiotic treatments. For each antibiotic (ibezapolstat, fidaxomicin, vancomycin, and metronidazole), comparisons were made with baseline 2 (D14). Comparisons between D7 and D14 to account for changes in diversity due to dietary changes were performed using baseline 1 (D7) as the reference point. Significance symbols were determined based on the following p-value parameters: approximately 0 = "***", approximately 0.001 = "**", approximately 0.01 = "*", and approximately 0.05 = ".". Any p-values ​​>0.05 were not considered significant.

[0145] [Table 7]

[0146] Figures 9A-9E provide bar graphs of OTUs identified by random forest analysis that distinguish specific treatment groups. The dashed line represents the significance cutoff based on median importance given one standard deviation in both directions, and the OTUs shown (x-axis) are categorized at the family level. Additionally, the comparative and enriched (elevated) OTUs indicated in the legend are colored for each panel. Figures 9A-D show a comparison of each antibiotic-exposed mouse to the no-drug control group (ND control), while Figure 9E compares mice exposed to ibexapolstat with mice exposed to fidaxomicin.

[0147] At the phylum level, a significant proportional increase in Bacteroidetes was observed in the IBZ group, whereas an increase in Firmicutes was observed in the FDX group. Furthermore, the relative abundance of Proteobacteria and Verrucomicrobiota increased in the VAN and MTZ groups.

[0148] conclusion These studies show that a dietary change from pelleted to powdered chow reduces alpha diversity, causing a slight decrease in Bacteroidetes abundance and an increase in Verrucomicrobia (Akkermansia). All antibiotics perturbed the gut microbiome, but the changes appear to be drug-dependent. In general, ibexapolstat and fidaxomicin caused a proportional increase in Bacteroidetes, while vancomycin and metronidazole caused a proportional increase in Proteobacteria.

[0149] Furthermore, both IBZ and fidaxomicin administered to human-microbiome GF mice resulted in favorable but unique changes to the gut microbiota compared with the well-known gut microbiome-disrupting agents vancomycin and metronidazole. These results support the continued clinical development of IBZ for the treatment of CDI.

[0150] Example 7: Ibezapolstat modulates Clostridioides difficile virulence factors in vitro In Phase I and Phase IIa clinical trials conducted to date, the use of ibezazolstat has demonstrated favorable effects on the gut microbiome, which may predict anti-relapse pharmacological properties. Therefore, the purpose of this study was to evaluate the effects of IBZ on C. difficile virulence factors. To further this objective, this study investigated the in vitro effects of subinhibitory concentrations of IBZ on cell morphology, motility, and toxin production in C. difficile. Based on its potentially novel mechanism of action targeting the DNA pol IIIC enzyme, IBZ may demonstrate unique pharmacological properties beyond bacterial killing.

[0151] Methods and Materials C. difficile (C. difficile) reference strains CD630 and R20291 were treated with ibexapolstat at subinhibitory to minimally inhibitory concentrations. Toxin A and B concentrations were measured by ELISA (tgcBiomics). Expression of flagellar genes fliA, flgB, and fliC in CD630 was assessed after 4 h of treatment with IBZ using RT-qPCR. Morphological changes induced by IBZ treatment were assessed by bright-field microscopy at 10-40x magnification.

[0152] Morphological evaluation Cultures were prepared by inoculating one colony of C. difficile into brain heart infusion broth (BHI) supplemented with 0.1% sodium taurocholate. The cultures were then incubated in an anaerobic chamber at 37°C for 24 hours. The precultures were grown for approximately 10 days in fresh BHI containing 0.25x the MIC (0.5µg / mL) of the appropriate antibiotic. 6 After 24 hours of incubation, bright-field photographs were taken at 40x magnification using an EVOS imaging system (Thermofisher).

[0153] Motility assay C. difficile motility was assessed using a phenotypic motility assay. Cultures of bacterial strains were prepared by inoculating BHI medium containing 0.3% agar and a subinhibitory concentration of IBZ and grown anaerobically at 37°C for 48 hours. After incubation, bacterial strains were plated on semi-solid BHI agar for 76 hours and visually observed for movement.

[0154] Flagellar gene expression Bacterial precultures were diluted in BHI and grown for 4 h at 37°C under anaerobic conditions with subinhibitory concentrations of antibiotics. To assess flagellar gene expression, transcript levels of fliA, flgB, and fliC were measured by qRT-PCR (Doan et al. 2022).

[0155] Toxin production Bacterial precultures were diluted and then grown in BHI with subinhibitory concentrations of antibiotics for 24 h. Toxin production was assessed by ELISA (tgcBiomics) according to the manufacturer's instructions.

[0156] result In general, IBZ reduced toxin A and B concentrations in a dose-dependent manner. After normalizing toxin production to the control, a 55% (CD 630) to 60% (R20291) reduction in toxin levels was observed. Following 4 h of treatment with IBZ at 0.25× the MIC, a reduction of up to 50% in motility and flagellar genes was also observed. Finally, both CD 630 and R20291 strains exhibited an elongated cell phenotype at subinhibitory MICs of IBZ.

[0157] Morphological evaluation Figure 10 shows the effect of Ibezapolstat on CD630 morphology. As shown in Figure 10, IBZ exhibits a dose-dependent effect on C. difficile cell growth after 24 hours of treatment, affecting its cell division pathway. This phenotype has been previously observed with other antibiotics that target DNA.

[0158] Motility assay Figure 11 shows the effect of ibezazolstat on CD 630 motility. As can be seen in Figure 11, treatment of CD 630 with subinhibitory concentrations of ibezazolstat in semi-solid BHI agar for at least 48 hours (up to 76 hours) resulted in attenuation of motility.

[0159] Flagellar gene expression Figure 12 shows the effect of ibezazolstat on flagellar gene expression. After 4 hours of treatment with a subinhibitory concentration of IBZ (0.5x the MIC), CD630 showed a 2- to 5-fold reduction in flagellar gene expression compared to the gluD control (Figure 12).

[0160] Toxin production Figure 13 shows the effect of ibezazolstat on modulating C. difficile toxin production. As Figure 13 demonstrates, IBZ reduces total toxin A and B production in strain CD 630 in a dose-dependent manner after 24 hours of treatment.

[0161] conclusion As shown in Figures 10-13, ibezazolstat treatment demonstrates in vitro effects on C. difficile virulence determinants. At subinhibitory levels, ibezazolstat affects several aspects of C. difficile virulence. For example, IBZ influences bacterial fitness through modulation of morphology, resulting in impaired cell division. IBZ also reduces C. difficile motility, as visualized in the motility assay shown in Figure 11. Furthermore, IBZ attenuated the relative expression of flagellar genes fliA, flgB, and fliC, reducing C. difficile production of both toxin A and toxin B. These preliminary results elucidate the mechanism of action of IBZ and support its continued clinical development.

[0162] Example 8: In vitro biofilm studies of Ibezapolstat and comparative antibiotics against C. difficile The high recurrence rate of Clostridioides difficile infection (CDI) is hypothesized to be partially attributable to biofilm formation. IBZ, a novel Pol IIIC DNA polymerase inhibitor antibiotic, has been shown to have a low CDI recurrence rate in a Phase II clinical trial; however, its in vitro effect on C. difficile biofilms is unknown. Therefore, the purpose of this study was to compare IBZs' C. difficile antibiofilm activity with comparable antibiotics.

[0163] Methods and Materials Antimicrobial activity and biofilm biomass studies were used to compare the effects of IBZ and comparable antibiotics on C. difficile biofilms. These studies compared IBZ with the antimicrobial agents vancomycin (VAN), fidaxomicin (FDX), and metronidazole (MTZ). A basic flowchart outlining the experimental procedures used in these studies is shown in Figure 14.

[0164] Biofilm formation Briefly, C. difficile laboratory strain R20291 was grown in brain heart infusion supplement (BHIS) medium. To initiate biofilm cultures, 100 μL of C. diff. culture (OD = 0.01) was added to each well of a 96-well plate. Biofilm formation was established by anaerobic incubation of the cultures at 37°C for 24 hours. Early biofilm formation was established at 4 hours, whereas late biofilm formation was established at 24, 48, and 72 hours.

[0165] Antibacterial activity After biofilm formation, the BHIS medium was removed and replaced with fresh medium without antibiotics (control), ibezazolstat (IBZ), fidaxomicin (FDX), or metronidazole (MTZ). Cultures were then incubated anaerobically for 24 h or subjected to a time-course study.

[0166] Biofilm biomass Biofilm biomass was measured using crystal violet staining. Cultures were stained with 0.2% crystal violet for 30 minutes. Data were measured and recorded using a Cytation 3 calibrated to read at A570. Data were analyzed by comparing the percent growth of antibiotic-treated cultures with the control.

[0167] result Figure 15 shows early (24 h) and late (>72 h) biofilm formation in biofilms embedded with either the R20291 or CD 630 C. diff strains. Figures 17A and 17B demonstrate that IBZ and vancomycin (VAN) inhibit C. diff biofilm formation and reduce C. diff biofilm biomass at multiple time points. Compared to controls, IBZ and VAN reduced C. diff growth (CFU / mL) and biomass in early biofilms at sub-MIC (0.4×MIC) and above the MIC of VAN (40×MIC), whereas IBZ eradicated growth after 48 h of treatment (see Figures 17A and 17B).

[0168] Antibacterial activity Figure 16 shows 24-hour biofilms embedded with either the R20291 or CD 630 C. diff strains. As can be seen in Figure 16, the minimum bactericidal concentrations were similar for vegetative non-biofilm growth for all four antibiotics, although an Eagle effect was observed at higher concentrations of MTZ.

[0169] Biofilm biomass As noted above, Figures 17A and 17B show the effects of IBZ and VAN on early biofilms (i.e., biofilms formed at 4 hours). As seen in Figure 17A, both IBZ and VAN, at MIC and sub-MIC levels, reduced biofilm growth within 48 hours as measured by CFU / mL. Furthermore, Figure 18 shows that late-stage biofilms (i.e., biofilms formed over 24 hours) embedded with R20291 or CD630 exhibited biphasic growth with reduced biomass at 48 and 24 hours, with regrowth observed at 72 hours (likely due to biofilm detachment). As shown in Figure 18, although differences were observed, all antibiotics reduced biofilm biomass compared to the control, regardless of biofilm growth time, with the highest reduction observed during later biofilm growth periods (48 and 72 hours). Additionally, Table 8 provides the MICs and MBICs of the tested antibiotics in μg / mL applicable to C. diff. strain R20291. [Table 8]

[0170] conclusion These studies demonstrate that IBZ can be as effective as comparable antibiotics in reducing both the amount of C. difficile embedded in biofilms and the biomass of the biofilm. These results, along with the success of clinical trials to date, warrant the continued development of IBZ.

[0171] Other embodiments Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention. Various substitutions, changes, and modifications can be made to the invention without departing from the spirit and scope of the invention. Other aspects, advantages, and modifications are within the scope of the invention. The contents of all references, issued patents, and published patent applications cited throughout this application are incorporated herein by reference. The appropriate components, processes, and methods of these patents, applications, and other documents may be selected for the present invention and its embodiments.

[0172] References 1. Mullish BH, Quraishi MN, Segal JP, et al., The gut microbiome: what every gastroenterologist needs to knowFrontline Gastroenterology 2021;12:118-127. 2. Kachrimanidou M, Tsintarakis E. Insights into the Role of Human Gut Microbiota in Clostridioides difficile Infection. Microorganisms. 2020; 8(2):200. https: / / doi.org / 10.3390 / microorganisms8020200. 3. Mueller NT, et al., The infant microbiome development: mom matters. Trends Mol Med. 2015 Feb;21(2):109-17. doi: 10.1016 / j.molmed.2014.12.002. Epub 2014 Dec 11. PMID: 25578246; PMCID: PMC4464665. 4. Jangi, S. and Lamont, T., (2010) Asymptomatic Colonization by Clostridium difficile in Infants: Implications for Disease in Later Life, Journal of Pediatric Gastroenterology and Nutrition. 51(1):2-7. 5. Huemer, M. et al., Antibiotic resistance and persistence-Implications for human health and treatment perspectives, EMBO Rep. 2020 Dec 3; 21(12): e51034. 6. Tanwar J, Das S, Fatima Z, Hameed S (2014) Multidrug resistance: an emerging crisis. Interdiscip Perspect Infect Dis 2014: 541340. 7. Spellberg B, Bartlett JG, Gilbert DN (2013) The future of antibiotics and resistance. N Engl J Med 368: 299-302. 8. Rice LB (2010) Progress and challenges in implementing the research on ESKAPE pathogens. Infect Control Hosp Epidemiol 31(Suppl 1): S7-10. 9. Magill, et al., “Changes in Prevalence of Health Care-Associated Infections in U.S. Hospitals” The New England Journal of Medicine 2018, 379, 1732-1744. 10. Li, W., et al., (2022), Evaluation of culturable 'last-resort' antibiotic resistant pathogens in hospital wastewater and implications on the risks of nosocomial antimicrobial resistance prevalence. Journal of hazardous materials, 438, 129477. https: / / doi.org / 10.1016 / j.jhazmat.2022.129477 11. Davis ML, et al. Multicentre derivation and validation of a simple predictive index for healthcare-associated C. difficile infection. (Clin Microbiol Infect 2018; 24: 1190-4). 12. Chambers HF, Deleo FR (2009) Waves of resistance: Staphylococcus aureus in the antibiotic era. Nat Rev Microbiol 7: 629-641. 13. Isaac S, et al., Short- and long-term effects of oral vancomycin on the human intestinal microbiota. J Antimicrob Chemother 2017; 72: 128-36. 14. Peng Z, et al., Update on antimicrobial resistance in C. difficile: resistance mechanisms and antimicrobial susceptibility testing. (J Clin Microbiol 2017; 55: 1998-2008). 15. Garey KW, et al. A randomized, double-blind, placebo-controlled, single and multiple ascending dose Phase 1 study to determine the safety, pharmacokinetics and food and faecal microbiome effects of ibezapolstat administered orally to healthy subjects. J Antimicrob Chemother 2020;75(12):3635-3643. DOI: 10.1093 / jac / dkaa364. 16. Gonzales-Luna AJ, Carlson TJ, Dotson KM, et al. PCR ribotypes of Clostridioides difficile across Texas from 2011 to 2018 including emergence of ribotype 255. Emerg Microbes Infect 2020; 9(1): 341-7. 17. Kornberg, et al., “DNA replication” 1992, W. H. Freeman & Co., New York, N.Y. 18. Tarantino, et al., “Inhibitors of DNA polymerase III as novel antimicrobial agents against Gram-positive eubacteria” Antimicr. Agents Chemother. 1999, 43, 1982-1987. 19. Begum K, Basseres E, Miranda J, et al. In Vitro Activity of Omadacycline, a New Tetracycline Analog, and Comparators against Clostridioides difficile. Antimicrob Agents Chemother 2020; 64(8). Other References Wright, et al., “Inhibition of Bacillus subtilis DNA Polymerase III by Arylhydrazinopyrimidine” Biochimica et Biophysica Acta 1976, 432, 37-48 Wright, et al., “DNA polymerase III: A new target for antibiotic development”, Current Op. Anti- Infective Investigat. Drugs 1999, 1, 45-48. Tarantino, et al., “6-Anilinouracil-Based Inhibitors of Bacillus subtilis DNA Polymerase III: Antipolymerase and Antimicrobial Structure-Activity Relationships Based on Substitution at Uracil N3” J. Med. Chem. 1999, 42, 2035-2040. Daly, et al., “In vitro antimicrobial activity of novel anilinouracils which selectively inhibit DNA polymerase III of Gram-positive bacteria” Antimicr. Agents Chemother. 2000, 44, 2217-2221. Ali, et al., “Design and Synthesis of Novel Antibacterial Agents with Inhibitory Activity against DNA Polymerase III” Bioorg. Med. Chem. Lett. 2001, 11, 2185-2188. Muto, et al., “SHEA Guideline for Preventing Nosocomial Transmission of Multidrug-Resistant Strains of Staphylococcus aureus and Enterococcus” Infection Control and Hospital Epidemiology 2003, 362-386. Ali, et al., “Novel Pyrazolo [3,4-d]pyrimidine-Based Inhibitors of Staphylococcus aureus DNA Polymerase III: Design, Synthesis, and Biological Evaluation” J. Med. Chem. 2003, 46, 1824-1830. Zhi, et al., “Synthesis and Antibacterial Activity of 3-Substituted-6-(3-ethyl-4-methylanilino) uracils” J. Med. Chem. 2005, 48, 7063-7074. Wright. et al., “Active site directed inhibitors of replication-specific bacterial DNA polymerases” Bioorg Med Chem Lett. 2005, 15, 729-732. Kuhl, et al., “Biological Characterization of Novel Inhibitors of the Gram-Positive DNA Polymerase IIIC Enzyme” Antimicr. Agents Chemother. 2005, 49, 987-995. Wright, et al., “Purine and Isosteric Antibacterial Compounds“ US Patent: 6,926,763 (2005) Zhi, et al., “Hybrid Antibacterials. DNA polymerase topoisomerase inhibitors.” J. Med. Chem. 2006, 49, 1455-1465. Evans, et al., “Structure of Po / C reveals unique DNA binding and fidelity determinants” Proc Natl Acad Sci U S A 2008, 105, 20695-700. Cer, et al., “IC 50 -to-Ki: a web-based tool for converting IC 50 to Ki values for inhibitors of enzyme activity and ligand binding” Nucleic Acids Research, 2009, Vol. 37, Web Server issue W441-W445 doi:10.1093 / nar / gkp253 Hookman. et al., “Clostridium difficile associated infection, diarrhea and colitis,” World J. Gastroenterol 2009, 15, 1554-1580. Torti, et al., “Clostridium difficile DNA polymerase IIIC: Basis for activity of Anti-Bacterial Compounds” Current Enzyme Inhibition 2011, 7, 147-153. Xu, et al., “7-Alkyl-N2-substituted-3-deazaguanines. Synthesis, DNA polymerase III inhibition and antibacterial activity” Bioorg Med Chem Lett. 2011, 21, 4197-202. Dvoskin, et al., “A novel agent effective against infection with Clostridium difficile” Antimicr. Agents Chemother. 2012, 56, 1624-1626. Guile, et al., “Antibacterial sulfone and sulfoxide substituted heterocyclic urea compounds” US Patent: 8,293,919 (2012), Guile, et al., “Antibacterial heterocyclic ureas” US Patent: 8,716,320 (2014). Wright, et al., “Selective Antibacterials for Clostridium difficile Infections” US Patent: 8,796,292 (2014). Schloss, P.D., et al., Introducing mothur: open-source, platform-independent, community supported software for describing and comparing microbial communities. Appl Environ Microbiol, 2009. 75(23): p. 7537-41. Kozich, J.J., et al., Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Appl Environ Microbiol, 2013. 79(17): p. 5112-20.). R Core Team (2023). R: A Language and Environment for Statistical Computing, Vienna, Austria.).

Claims

1. 1. A method of promoting gut microbiome health in a subject, comprising: administering an effective amount of a low G+C preferential nucleoside (LDN) analog to a subject suffering from said intestinal dysbiosis; The LDN analog maintains and / or increases beneficial microorganisms in the intestinal microbiome, while simultaneously reducing harmful gram-positive bacteria whose genomes contain low guanine and cytosine content (low G + C) in the intestinal microbiome.

2. 2. The method of claim 1, wherein the LDN analog is a small molecule inhibitor of DNA pol IIIC enzyme.

3. 3. The method of claim 2, wherein the LDN analogue is targeted to the DNA pol IIIC of low G+C class bacteria whose genome contains less than 50% guanine (G) + cytosine (C).

4. The method of claim 1, wherein the LDN analogue selectively targets physiologically harmful species belonging to Streptococcus, Enterococcus, Staphylococcus, Bacillus, Clostridioides, Pneumococcus, Listeria, Mycoplasma and / or Lactobacillus.

5. 10. The method of claim 1, wherein the beneficial microorganisms comprise the phylum Firmicutes, including the family Lachnospiraceae and Lactobacillaceae.

6. 6. The method of claim 5, wherein administering the effective amount of the LDN analog reduces or prevents the re-growth of low G+C content Gram-positive pathogenic commensals in the intestinal microbiome within 30 days.

7. 1. A method of achieving and / or maintaining a healthy proportion of gut microflora in the gut environment of a subject, comprising: administering to the subject an effective amount of a compound against a DNA pol IIIC enzyme in a low G+C content Gram-positive pathogenic commensal organism; Reducing or eliminating physiologically harmful pathogenic microorganisms belonging to the phylum Bacillus; and increasing and / or maintaining physiologically beneficial microorganisms in the intestinal environment.

8. 8. The method of claim 7, wherein the compound is a low G+C-directed nucleoside (LDN) analog.

9. 9. The method of claim 8, wherein the physiologically harmful pathogenic microorganisms comprise species belonging to the phylum Firmicutes and / or the order Bacillales.

10. 9. The method of claim 8, wherein the physiologically beneficial microorganisms in the intestinal environment comprise members of the phylum Actinomycetota, including Lachnospiraceae and / or Lactobacillaceae.

11. 9. The method of claim 8, wherein the administration of the LDN analog is prophylactic, the subject is healthy, and the LDN analog restores or maintains a symbiotic relationship between the subject and microorganisms in the intestinal environment.

12. 8. The method of claim 7, wherein the proportion of bacterial phyla in the subject's intestinal microbiome is adjusted to a healthier balance compared to the subject's intestinal microbiome before administration of the LDN analog.

13. 9. The method of claim 8, wherein the physiologically beneficial microorganisms in the intestinal environment comprise anaerobic Gram-positive bacteria belonging to the genus Clostridium, including C. coccoides.

14. 13. The method of claim 12, wherein the subject is suffering from an overgrowth of Clostridioides difficile in the intestine.

15. 12. The method of claim 11, wherein administering the LDN analogue is continued until the majority of bacterial species are from the phylum Actinobacteria, Firmicutes or Bacteroidetes, and the minority are from the phylum Proteobacteria.

16. A composition for promoting gut microbiome health comprising a low G+C-directed nucleoside (LDN) analog, wherein the LDN analog inhibits DNA pol IIIC enzymes in physiologically harmful pathogenic commensals, thereby reducing harmful Gram-positive bacteria while promoting the proliferation of beneficial microorganisms in the gut microbiome.

17. 17. The composition of claim 16, wherein the LDN analog is a preventative treatment and promotes the persistence and / or repopulation of a healthy microbiota.

18. 17. The composition of claim 16, wherein the subject receives at least 450 mg of the LDN analog at least once a day.

19. 17. The composition of claim 16, wherein the LDN analog selectively reduces the growth of pathogenic members of Streptococcus, Enterococcus, Staphylococcus, Bacillus, Clostridioides, Pneumococcus, Listeria, Mycoplasma and / or Lactobacillus.

20. A composition for reducing physiologically harmful Gram-positive organisms in the intestinal microbiome, the composition comprising a low G+C-directed nucleoside (LDN) analog, wherein the LDN analog inhibits the motility of the Gram-positive organisms.

21. 21. The composition of claim 20, wherein the LDN analog inhibits the motility of the Gram-positive organism by reducing flagellar gene expression.

Images