Compounds and methods for improving health

Abstract

The present technical field relates to methods for promoting intestinal microbiome health using low G + C directed (LDN) analogs. More specifically, the field of technology relates to a method for selectively reducing physiologically harmful gram-positive bacteria 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, the genome of the Gram-positive bacterium has a low guanine and cytosine content (actually, the genome comprises greater amounts of adenine and thymine / uracil nucleotides).

Description

[0001] All publications, patents, and patent applications mentioned herein are incorporated herein 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. Technical Field

[0002] This technical field relates to methods for promoting gut microbiome health using low G+C directed (LDN) analogs. More specifically, this technical field relates to methods of using LDN analogs as inhibitors of DNA polymerase IIIC (DNA pol IIIC) enzymes to selectively reduce physiologically harmful Gram-positive bacteria in the gut microbiome while maintaining or increasing the proportion of beneficial microbial communities, the genomes of which Gram-positive bacteria have a low guanine and cytosine content (in fact, the genomes contain a greater amount of adenine and thymine / uracil nucleotides). Background Art

[0003] The mucosal surfaces of the body contain complex and specialized microbial communities, commonly 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). It is estimated that the microbiome of the human gastrointestinal tract consists of up to 100 trillion microorganisms, the majority 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). Although the gastrointestinal microbiome is highly diverse, in healthy adults, it is predominantly composed of bacteria from two major phyla, namely Firmicutes (spore-forming Gram-positive organisms) and Bacteroidetes (non-spore-forming Gram-negative organisms). These two phyla typically account for 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 Firmicutes and Bacteroidetes, the gut microbiome is also composed of other 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). Proteobacteria are composed of Gram-negative facultative anaerobes. Although some members of Proteobacteria that are present in small amounts are part of a healthy gut, this phylum also contains common unwanted Gram-negative pathobionts, 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 a large proportion in children and the overall proportion generally decreases with age (replaced by Firmicutes and Bacteroidetes). At birth, facultative anaerobic species such as Escherichia coli, Staphylococcus, and Streptococcus colonize the infant gut and create an anaerobic environment within the first few days of life, which allows strict anaerobes (anaerobes that cannot grow in the presence of dissolved oxygen exceeding 5 μM), such as Bacteroides (Bacteroidetes) and Bifidobacterium (Actinobacteria), to proliferate (Mueller NT, et al., The infant microbiome development: mom matters. Trends Mol Med. February 2015;21(2):109–17. doi:10.1016 / j.molmed.2014.12.002. Epub December 11, 2014. PMID:25578246; PMCID:PMC4464665). During the first year of life, and through the infant's exposure to the environment and breast milk or formula, the gut microbiome evolves into a mature biome similar to the adult gut microbiome. (Jangi S, 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 has a symbiotic relationship with the host. Through this mutualistic relationship, the microbiome provides many benefits to the host, including shaping the gut and systemic immune systems, maintaining a healthy gut epithelium, extracting 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 Gastroenterol. 2021;12:118-127). When the composition of the microbiome changes from its normal diversity, these beneficial physiological functions are disrupted. This is known as dysbiosis. (Mullish BH, Quraishi MN, Segal JP, et al. The gut microbiome: what every gastroenterologist needs to know. Frontline Gastroenterol. 2021;12:118-127). When the gut microbiome is in a state of dysbiosis, there are fewer beneficial microorganisms (commensals) in the microbiome and more potentially harmful microorganisms (pathogens). (Mullish BH, Quraishi MN, Segal JP, et al. The gut microbiome: what every gastroenterologist needs to know. Frontline Gastroenterol. 2021;12:118-127).

[0007] Of crucial importance to health are anaerobic bacteria, i.e., bacteria that grow in an oxygen-free atmosphere, such as those found in the intestinal environment. Gram-positive anaerobic bacteria, such as Lactobacilli, Bifidobacteria, and Eubacteria, as well as Gram-negative anaerobic bacteria, such as Bacteroides, represent "beneficial" gut organisms that are crucial to health. In contrast, the Gram-positive anaerobic bacteria Clostridioides difficile and Clostridioides perfringens represent pathogenic bacteria. In particular, C. diff. has an increasingly strong association with patient illness, possibly due to patients' reliance on antimicrobial drugs to treat pathogenic bacterial infections.

[0008] The emergence and use of antibiotic drugs have shaped modern medicine. The mid-20th century was even named the "antibiotic era". In fact, it was thought that infectious diseases had 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., December 3, 2020; 21(12):e51034). Although antibiotics are essential in treating and curing a range of bacterial infections, an increasing number of bacteria are becoming resistant to an increasing number of the antibiotics currently in use. This widespread use of antimicrobial agents 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). Due to the scarcity of new antimicrobial drugs and the increasing prevalence of MDR bacteria, resulting in treatment failure, antibiotic-resistant bacteria have become a major threat to modern healthcare (Spellberg, B, Bartlett, JG, Gilbert, DN (2013) The future of antibiotics and resistance. N Engl J Med 368:299-302). Thus, bacterial pathogens pose a serious threat to public health.

[0009] Nosocomial pathogens and infections are pathogens and infections that are acquired within a hospital and pose a considerable threat to hospital staff and patients. Hospital pathogenic bacteria with increasing multidrug resistance and toxicity are known as ESKAPE pathogens (Huemer, M. et al., Antibiotic resistance and persistence - implications for human health and therapeutic prospects, EMBO Reports, 3 December 2020; 21(12):e51034). ESKAPE represents the Gram-positive bacterial species Enterococcus faecium and Staphylococcus aureus, as well as the Gram-negative bacteria Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. ESKAPE pathogens impose a heavy burden on patient health and the healthcare system. These pathogens are characterized by a high degree of MDR and are the cause of nosocomial-acquired and potentially life-threatening infections in critically ill and immunocompromised patients (Huemer, M. et al., Antibiotic resistance and persistence - implications for human health and therapeutic prospects, EMBO Reports, 3 December 2020; 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 the resistance of microorganisms to many antimicrobial drugs continues to increase, the use of "last resort" antibiotics such as tigecycline, polymyxin E, daptomycin, vancomycin, and linezolid has become increasingly 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, 129477). These so-called last resort antibiotics serve as the "last line of defense" against infections by antibiotic-resistant pathogens. Therefore, the increasing prevalence of 'last resort' antibiotic-resistant pathogens in the hospital environment and the nosocomial transmission of these pathogens pose a serious threat to the health of patients (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 faecalis / faecium, are the major nosocomial pathogens; together they account for most current nosocomial diseases. In addition, Clostridioides difficile infection (CDI) is the most common healthcare-associated infection in the United States. (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). Importantly, Clostridioides difficile may sometimes be a normal component of the healthy gut microbiome. However, during microbiome dysbiosis (which may be the result of antibiotic treatment), Clostridioides difficile may proliferate and cause disease, CDI).

[0011] Overgrowth of C. difficile in the intestinal environment can lead to a range of clinical symptoms, from mild diarrhea to severe life-threatening colon perforation and toxic megacolon (extreme inflammation and dilation of the colon). The use of last-resort antibiotics disrupts the host's microbiome, allowing CDI 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, through broad-spectrum antibiotic treatment, the phylum Bacteroidetes almost completely disappears, the phylum Firmicutes decreases, and the phylum Proteobacteria overgrows; these changes allow C. difficile spores to germinate, leading to increased growth and an increased likelihood of pathogenesis (Mullish BH, Quraishi MN, Segal JP, et al., The intestinal microbiome: what every gastroenterologist needs to know. Frontline Gastroenterol 2021;12:118-127). Despite the risk of antimicrobial resistance and due to limited treatment options, antimicrobial therapy remains the first line of defense against pathogenic microbial infections. For example, antibiotic treatment remains the mainstay for treating pathogens such as Staphylococcus aureus, C. difficile, Enterococcus faecalis / faecium, and other known bacterial pathogens.

[0012] Compared to Gram-negative bacteria, which have low permeability to many antimicrobial compounds (e.g., vancomycin), the Gram-positive pathobiont Staphylococcus aureus is naturally sensitive to almost every developed antibiotic. Despite being sensitive to antimicrobials, it is well known that S. aureus and similar pathobionts quickly acquire antibiotic resistance. Typically, such pathobionts acquire antimicrobial resistance by obtaining specific genetic modifications, such as acquiring favorable mutations or undergoing horizontal gene transfer. Due to the iterative relationship between antimicrobial development and pathobiont evolution, these types of pathogenic bacterial infections tend to occur in waves of epidemics (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 in the treatment guidelines of the Infectious Disease Society of America (IDSA); however, vancomycin has been associated with a high recurrence rate of CDI and has been shown to have 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] In addition, treatment with antibacterial compounds such as vancomycin may lead to a decrease in the microbiome diversity of Firmicutes, Actinobacteria, and Bacteroidetes, accompanied by overgrowth of characteristic Proteobacteria. (KW Garey, 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. Journal of Antimicrobial Chemotherapy 2020;75(12):3635-3643). Overgrowth of Proteobacteria is significantly associated with a substantially increased risk of systemic infection with MDR Gram-negative microorganisms. Treatment with vancomycin for intestinal bacterial infections is associated with high recurrence rates and dysbiosis, with approximately 20-25% of patients experiencing recurrence of infection after discontinuation of treatment. (AJ Gonzales-Luna, TJ Carlson, KM Dotson, et al., PCR ribotypes of Clostridioides difficile across Texas from 2011 to 2018 including emergence of ribotype 255. Emerging Microbes & Infections 2020;9(1):341-7). Therefore, there is an urgent need for new therapies with unique mechanisms of action against harmful bacterial pathogens.

[0014] In particular, there is a need for compounds that can reduce or eliminate the overgrowth of harmful microorganisms in the intestinal environment that may cause infection or disease, while also maintaining the delicate balance of the microorganisms that make up the gut microbiome. Such treatments may lead to clinical cure and also prevent recurrent overgrowth, infection, and / or dysbiosis. Therefore, there is also a need for treatments that promote microbiome health. 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 an individual.

[0016] The present disclosure provides methods for promoting gut microbiome health in an individual, comprising: administering to an individual suffering from gut dysbiosis an effective amount of a low G+C directed nucleoside (LDN) analog; wherein the LDN analog simultaneously reduces or eliminates the growth of harmful Gram-positive bacteria having a low G+C content in the gut microbiome while maintaining and / or increasing beneficial microorganisms in the gut microbiome. In some aspects, the LDN analog can be a small molecule inhibitor of the DNApol IIIC enzyme, such as ibezapolstat. In some aspects, the LDN analog can target the DNApol IIIC of low G+C bacteria whose genomes contain <50% guanine (G) + cytosine (C). In some aspects, the LDN analog can selectively target physiologically harmful species belonging to Streptococcus, Enterococcus, Staphylococcus, Bacillus, Clostridioides, Pneumococcus, Listeria, Mycoplasma, and / or Lactobacillus. In some aspects, the beneficial microorganisms can comprise Firmicutes and include Lachnospiraceae and Lactobacillaceae. In other aspects, administering an effective amount of the LDN analog can reduce the growth or prevent the regrowth of Gram-positive pathogenic bacteria with low G+C content in the gut microbiome within 30 days.

[0017] The present disclosure also discloses a method for achieving and / or maintaining a healthy proportion of the gut microbial community in the intestinal environment of an individual, comprising: administering to the individual an effective amount of a compound directed against the DNA pol IIIC enzyme in Gram-positive pathogenic bacteria with low G+C content; reducing or eliminating physiologically harmful pathogenic microorganisms belonging to the phylum Bacillus; and increasing and / or maintaining physiologically beneficial microorganisms in the intestinal environment. In some instances, the compound can be an LDN analog. In some aspects, administering the LDN analog can be prophylactic, the individual can be healthy, and the LDN analog can restore or maintain the mutualistic symbiotic relationship between the individual and the microorganisms in the intestinal environment. Alternatively or additionally, in some aspects, the individual's gut may be suffering from an overgrowth of Clostridioides difficile. In some instances, the physiologically beneficial microorganisms in the intestinal environment can comprise anaerobic Gram-positive bacteria belonging to the genus Clostridium and include Clostridium coccoides.

[0018] The present disclosure also provides a composition for promoting gut microbiome health, which comprises a low G+C directed nucleoside (LDN) analogue, wherein the LDN analogue inhibits the DNA polIIIC enzyme in physiologically harmful pathogenic bacteria, thereby reducing harmful Gram-positive bacteria while allowing beneficial microorganisms to proliferate in the gut microbiome. Additionally, in some aspects, the LDN analogue can be a prophylactic treatment and can promote the persistence and / or regrowth of a healthy microbiota. In some examples, the LDN analogue can selectively reduce the growth of pathogenic members of Streptococcus, Enterococcus, Staphylococcus, Bacillus, Clostridium, Pneumococcus, Listeria, Mycoplasma, and / or Lactobacillus. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] This patent or application file contains at least one color drawing. Copies of this patent or patent application publication with one or more color drawings will be provided by the Patent Office upon request and payment of the necessary fees.

[0020] The drawings described herein are for illustrative purposes only and do not limit the scope of the disclosure in any way.

[0021] Figure 1 presents the results of a qPCR experiment. Figure 1A Shows the quantification of relevant Firmicutes isolated from samples taken from healthy volunteers in Phase 1; Figure 1B Shows the quantification of relevant Firmicutes isolated from samples taken from individuals with CDI in Phase 2a;

[0022] Figure 2 is a schematic diagram showing the sensitivity of Firmicutes to ibezapolstat (IBZ). Figure 2A Shows variable sensitivities between Clostridium butyricum strains (Clostridium butyricum 1008 and Clostridium butyricum 1007); and Figure 2B Is a description and comparison of the structures of two Clostridium butyricum strains;

[0023] Figure 3 Is a graph that shows on the x-axis targeted flagellar genes and on the y-axis the relative expression of these genes compared to an untreated control. Additionally, to visualize the data more vividly, the relative expression of the control is normalized to a value of 1;

[0024] Figure 4 Presents a motility assay that compares the motility of a control strain of Clostridium difficile with a strain obtained from an isolate and exposed to a sub-inhibitory (non-killing) concentration of ibezapolstat;

[0025] Figure 5Shows a figure that depicts the changes in the gut microbiome after antibiotics in patients with Clostridioides difficile infection (CDI) and how this increases the chance of CDI recurrence. The figure was designed using BioRender;

[0026] Figure 6 Shows a figure depicting the study design of germ-free humanized mice. The figure was created using BioRender;

[0027] Figure 7A -E presents a box plot with groups connected by lines based on color. The color indicates the antibiotic to which the mice were exposed during the last 10 days of the experiment (or the lack of antibiotic in the case of the no-drug control (ND control) and baseline samples). Figure 7A and 7B A graph depicting the changes in the α-diversity of the gut microbiome throughout the experiment. Figure 7A Shows the changes in the richness of operational taxonomic units (OTUs) within the gut microbiome during the experiment; and Figure 7B Shows the changes in the inverse-Simpson's Index within the gut microbiome during the experiment. Figure 7C 、 7D and 7E present a graph depicting the changes in the β-diversity (diversity between groups) of the gut microbiome throughout the experiment. Figure 7C Shows the changes in the β-dispersion (distance to the centroid) of the gut microbiome throughout the experiment; Figure 7D Shows the changes in the Bray-Curtis Dissimilarity to baseline 1 (day 7) during the experiment; and Figure 7E Shows the changes in the Bray-Curtis Dissimilarity to baseline 2 (day 14); and Figure 7F Shows the non-metric multidimensional scaling analysis (NMDS) of the Bray-Curtis Dissimilarity. Each point represents a sample taken at the corresponding time point (marked at the top of each graph). The size indicates the inverse-Simpson's Index value of the sample, the shape indicates from which trial the sample was collected, and the color indicates which antibiotic (or lack of antibiotic) each sample was exposed to.

[0028] Figure 8A -D shows a stacked bar graph that illustrates the average relative abundances (expressed as percentages) of different bacterial taxonomic ranks throughout 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;

[0029] Figure 9A-D presents bar graphs of OTUs identified by random forest analysis that distinguish the designated treatment groups. The dashed line represents the significance cutoff based on the median importance level, assuming 1 standard deviation in both directions, and the OTUs shown (x-axis) are classified to the family level. For each group, the OTUs that are compared and enriched (elevated) as shown in the legend are colored. Figure 9A -D shows comparisons of each group of mice exposed to antibiotics or the no-drug control group (ND control). Figure 9E Compare mice exposed to ibezaprost with mice exposed to fidaxomicin;

[0030] Figure 10 Shows the effect of ibezaprost on the morphology of Clostridium difficile strain CD 630;

[0031] Figure 11 Shows the effect of ibezaprost on the motility of CD 630;

[0032] Figure 12 Shows the effect of ibezaprost on flagellar gene expression;

[0033] Figure 13 Shows the effect of ibezaprost on regulating Clostridium difficile toxin production;

[0034] Figure 14 Presents a basic flowchart outlining the experimental procedures used in these studies, which can be seen;

[0035] Figure 15 Shows the biofilm formation of Clostridium difficile laboratory strains R20291 and CD630;

[0036] Figure 16 Presents the minimum biofilm inhibitory concentration (MBIC) and Eagle effect corresponding to antibiotic treatment with IBZ, vancomycin (VAN), fidaxomicin (FDX), or metronidazole (MTZ);

[0037] Figure 17 Is a graph demonstrating the effect of the MIC or sub-MIC levels of IBZ and VAN on early (4-hour) biofilms by measuring CFU / mL over a 24- or 48-hour period;

[0038] Figure 18 Is a graph demonstrating the effect of the MIC or sub-MIC levels of IBZ and VAN on early (4-hour) biofilms by measuring the optical density at A570 over a 24- or 48-hour period;

[0039] Figure 19Shows the effects of IBZ and comparable antibiotics on early (24-hour) biofilms containing the laboratory strains of Clostridium difficile R20291 or CD630, and measures the optical density at A570 over a period of 24 or 48 hours. Detailed Description

[0040] The features and other details of the present invention will now be described more specifically. It should be understood that the specific embodiments described herein are shown by way of illustration and not as a limitation of the present invention. The main features of the present invention can be employed in various embodiments without departing from the scope of the present invention.

[0041] Methods and compositions for promoting gut microbiome health by administering low G+C directed nucleoside (LDN) analogs, such as DNA pol IIIC enzyme inhibitors, to an individual are described herein. The LDN analogs described herein can target low G+C bacteria, whose genomes contain <50% guanine (G) + cytosine (C). Additionally, the provided LDN analogs can selectively inhibit physiologically harmful pathogenic bacteria belonging to the phylum Bacillota, which can include members of the phylum Firmicutes or the order Bacillales. Further, the LDN analogs can inhibit the proliferation or replication of pathogenic bacteria from the genus Clostridium, such as Clostridium difficile, while also promoting the growth of beneficial members of the genus Clostridium, such as Clostridium globiforme. In some instances, the provided LDN analogs can selectively target Gram-positive members of the order Bacillales, including members of the family Staphylococcaceae and / or Enterococcus, such as Staphylococcus aureus and Enterococcus faecium. Additionally, the LDN analogs can promote the persistence or regrowth of a healthy microbiota in the intestinal environment. For example, in the case of administering the provided LDN analogs, the growth of the phylum Actinobacteriota in an individual's gut microbiome can increase. In some aspects, the LDN analogs can selectively target at least one single nucleotide polymorphism (SNP) in healthy microbiota organisms, thereby allowing these healthy organisms to continue to grow in the presence of LDN.

[0042] Methods and compositions that can target flagellar genes and thus cause a reduction in the motility of Gram-positive organisms are also described herein. For example, the provided compositions can cause a decrease in flagellar gene expression, including common flagellar-associated genes fliA, flgB, fliC-VIP.

[0043] Ibezapolstat (IBZ) is a non-absorbable antimicrobial agent for the treatment of Clostridioides difficile infection (CDI). In vitro and human studies have shown potent activity of IBZ against C. difficile, but selective activity against other beneficial Gram-positive gut microbiota shows a reduced risk of dysbiosis. Although the target DNA polIIIC enzyme is present in most Gram-positive species, IBZ sensitivity indicates selectivity among certain Gram-positive species found in the gut microbiota.

[0044] Definitions

[0045] Low G+C-directed nucleoside (LDN) analogues mean any molecule and / or compound in a class of nucleoside analogue inhibitors that can selectively target and inhibit DNA polymerase IIIC (DNA polIIIC) enzymes in Gram-positive microorganisms with a low G+C content (bacteria with a genome containing fewer guanine and cytosine nucleobases as opposed to adenine and thymine / uracil bases), such as those in the phylum Firmicutes and the order Bacillales with a low G+C content. Such molecules can include, but are not limited to, 7-substituted N2-(3,4-dichlorobenzyl)guanine (DCBG), and can selectively inhibit DNA polIIIC. Such molecules can also be 1,7-dihydro-6H-purin-6-one compounds, such as those in U.S. Pat. Nos. 6,926,763 and 8,796,292 incorporated herein by reference.

[0046] For example, ibezapolstat is 2-((3,4-dichlorobenzyl)amino)-7-(2-morpholinoethyl)-1,7-dihydro-6H-purin-6-one.

[0047] "Effective amount" means an amount sufficient to achieve a beneficial or desired clinical or biochemical result. The effective amount can be administered one or more times. For example, an effective amount can be an amount that, when administered to an infected or potentially infected site, will treat or prevent a bacterial infection while increasing the amount and / or proportion of Actinobacteria and / or Firmicutes in the microbiome.

[0048] "Selectively target" or "selectively targets" means a mechanism that specifically targets and inhibits the DNA polIIIC enzyme present in Gram-positive microorganisms with a low G+C content (where the genome has fewer guanine and cytosine nucleobases than adenine and thymine / uracil bases).

[0049] "Administration" or "administering" refers to the method of giving one or more unit doses of an LDN analogue, such as ibezaprost, to an animal (e.g., a mammal) (e.g., topical, oral, intravenous, intraperitoneal, or intramuscular administration). The method of administration can vary according to a variety of factors, such as the components of the pharmaceutical composition, the site of potential or actual infection, and the severity of the actual microbial infection.

[0050] "Inhibit" means reducing the cell growth rate of bacteria by at least 80%. In certain embodiments, growth can be inhibited by 90%, 95%, or even 99% or more. The degree of inhibition can be determined, for example, by in vitro growth assays, such as by standard liquid culture techniques. Colony formation is preferably inhibited at a suitable MIC (minimum inhibitory concentration), e.g., <100 μg / ml, more preferably <10 μg / ml.

[0051] "Treatment" refers to a method for obtaining a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in the degree of a disease, stabilization of a disease state (i.e., not getting worse), delay or slowing of disease progression, improvement or mitigation of a disease state, and remission (whether partial or complete), whether detectable or not. "Treatment" refers to both therapeutic treatment and prophylactic or preventive measures. Those in need of treatment include those who already have the disorder as well as those who want to prevent the disorder and / or prevent recurrence of the disorder. "Alleviating" a disease means a reduction in the degree of the disease state and / or the undesired clinical manifestations, and / or a slowing or prolongation of the course of progression compared to the situation without treatment.

[0052] "Microbiome" refers to the microorganisms in a particular environment (including the body or a part of the body). Preferably, the microbiome is located in the gut.

[0053] "Intestinal environment" refers to the internal environment of the intestinal system, which may have an intestinal microbiota and includes a complex community of bacteria, archaea, fungi, viruses, and protozoa that contribute more cells and genes to the host organism than their own.

[0054] A "healthy microbiome" can be described according to ecological stability (i.e., the ability to resist changes in community structure under stress or to rapidly return to baseline after stress-related changes), by an idealized (potentially health-related) composition, or by desired functional characteristics (including metabolism and nutrient supply to the host). The healthy adult microbiome is also characterized by the majority of bacterial species in the phyla Firmicutes or Bacteroidetes, as well as a minority of bacterial species in the phyla Actinobacteria and Proteobacteria. The healthy neonatal microbiome may be characterized by the majority of bacterial species in the phyla Bacteroidetes and Actinobacteria.

[0055] "Improving the health of the gut microbiome" means that most bacterial species in the composition of the microbiome are from the phylum Actinobacteria, Firmicutes or Bacteroidetes and a small portion are from the phylum Proteobacteria. Alternatively, improving the health of the gut microbiome may mean increasing the proportion of bacterial species in the phylum Actinobacteria, such as the proportion of bacterial species present in the gut microbiome of a healthy newborn. An individual may or may not have a pathogenic bacterial infection.

[0056] "Reducing the likelihood of infection" means a prophylactic treatment, or a treatment that, by administering a compound, causes a reduction (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90% or 95%) in the probability or rate of an individual or group of patients developing a microbial infection compared to an individual or group of patients not receiving an LDN analogue.

[0057] "Clinical cure" means that the initial infection has been cleared. It is preferably measured about 10 - 12 days after diagnosis after an individual has undergone a treatment course.

[0058] "Sustained clinical cure" means that an individual has achieved clinical cure and the infection has not recurred. It is measured 30 - 90 days after diagnosis.

[0059] "Relapse" means that an individual has achieved clinical cure and the infection recurs again within a period of 30 - 90 days.

[0060] "Animal" means any animal that is prone to Gram - positive bacterial infections. For example, animals can include humans, dogs, cats, pigs, cows, horses, goats, chickens, turkeys, sheep, rats, mice and rabbits, as well as other animals raised for commercial purposes or as pets. "Animals prone to microbial infections" are defined as animals with an increased risk of contracting a microbial infection relative to the general population. Examples of such animals include those who have recently undergone surgery, or immunocompromised individuals, such as those with AIDS (Acquired Immunodeficiency Syndrome) or transplant recipients who require immunosuppressive drugs. Such animals can be identified using methods known to those of ordinary skill in the art.

[0061] "Coating agent" is defined as a mixture of biocompatible compounds or terminating 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.

[0062] "Medium" is defined as any liquid or solid substance on or in which microorganisms may be present or the presence of which needs to be prevented. Exemplary media include culture media (e.g., agar or broth), food, medical supplies (e.g., sterile fluids), medical devices (e.g., catheters), work surfaces, and other surfaces.

[0063] "Microbial infection" is defined as the invasion of a host animal by pathogenic microorganisms. For example, an infection can include the overgrowth of microorganisms normally present in or on an animal or the growth of microorganisms 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 causes damage to a host animal. Thus, an animal "has" a microbial infection when an excessive microbial population is present in or on the animal or when the presence of the microbial population damages the cells or other tissues of the animal. In one embodiment, the number of a particular microbial genus or species is at least 2, 4, 6, or 8 times the number normally found in an animal. Examples of microorganisms include, but are not limited to, Gram-positive or any other class of bacteria.

[0064] "Pharmaceutically acceptable salt" means a salt derived from pharmaceutically acceptable inorganic and organic acids and inorganic and organic 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, p-toluenesulfonic acid, tartaric acid, acetic acid, citric acid, methane sulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Other acids such as oxalic acid, although not pharmaceutically acceptable per se, can be used as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts. Salts derived from appropriate bases include alkali metal (e.g., sodium or potassium) salts, alkaline earth metal (e.g., magnesium) salts, ammonium salts, and NR4 + (wherein R is C 1-4 alkyl) salts. Preferred salts include hydrochloride, hydrobromide, sulfate, mesylate, maleate, tartrate, and fumarate. Compounds according to the invention as referred to hereinafter include compounds of the general formula shown, as well as their pharmaceutically acceptable salts.

[0065] "Preventing" microbial growth or infection is defined as administering a compound of the invention to prevent microbial growth or infection from occurring. The amount of the compound of the invention required to prevent microbial growth can be determined, for example, by in vitro growth assays, such as by standard liquid culture techniques. The amount of the compound of the invention required to prevent microbial infection can be determined, for example, by in vivo assays, such as by determining the amount of the compound that must be administered to prevent infection in a study animal (e.g., guinea pig) after microbial inoculation. Generally, compounds that show prevention at suitable concentrations, e.g., <100 μg / ml, more preferably <10 μg / ml, can be used as therapeutic agents for further examination.

[0066] "Treatment" is defined as the medical management of a patient aimed at curing, ameliorating, or preventing a disease, pathologic condition, or disorder. The term includes active treatment, i.e., treatment specifically directed toward improving a disease, pathologic condition, or disorder, and also includes etiological treatment, i.e., treatment directed toward eliminating the cause of a disease, pathologic condition, or disorder. Additionally, the term includes palliative treatment, i.e., treatment aimed at alleviating symptoms rather than curing a disease, pathologic condition, or disorder; prophylactic treatment, i.e., treatment directed toward preventing a disease, pathologic condition, or disorder; and supportive treatment, i.e., treatment used to supplement another specific therapy directed toward improving a disease, pathologic condition, or disorder. The term "treatment" also includes symptomatic treatment, i.e., treatment directed toward the systemic symptoms of a disease, pathologic condition, or disorder.

[0067] "Therapeutically effective amount" is defined as an amount that, when administered to an animal in need thereof, will relieve at least some of the symptoms of a bacterial infection. In the case of prophylaxis, a "therapeutically effective amount" is an amount that, when administered to an animal susceptible to a bacterial infection, will help inhibit or otherwise reduce the likelihood of occurrence of such an infection.

[0068] "Prophylactic" or "prophylactic treatment" refers to the administration of an effective amount of an LDN analog to an individual who may be considered healthy, i.e., an individual who does not exhibit signs or symptoms of a bowel disease or dysbiosis. The individual may or may not be susceptible to intestinal dysbiosis and / or infection. For example, an LDN analog may be administered to an individual to maintain the microbial balance in a healthy gut, promote a mutually beneficial symbiotic relationship between the host gut and the microbes therein, and / or reduce the growth, replication, or proliferation of physiologically harmful pathogenic bacteria such that physiologically beneficial bacteria can thrive.

[0069] Details of one or more embodiments of the invention are set forth in the following accompanying description. Other features, objects, and advantages of the invention will be apparent from the specification and claims.

[0070] The microbiome of a healthy gut consists of major bacterial groups called phyla. Firmicutes (spore-forming Gram-positive organisms) and Bacteroidetes (non-spore-forming Gram-negative organisms) are the most common and together typically account for more than 90% of the healthy adult gut microbiome. The adult gut microbiome also contains Actinobacteria, Fusobacteria, Verrucomicrobia, and Proteobacteria. Actinobacteria are present in large proportions in children and their overall proportion generally decreases with age (being replaced by Firmicutes and Actinobacteria). Proteobacteria (Gram-negative facultative anaerobes) typically account for 2-5% of the healthy microbiome. When the composition of the microbiome changes from its normal diversity, normal physiological functions are disrupted, which is called dysbiosis. Patients with bacterial infections, such as Clostridioides difficile infection, are in a state of dysbiosis. Typically, dysbiosis associated with individuals with bacterial infections includes an increased proportion of Proteobacteria (commonly referred to as a "bloom") and a decrease in the numbers of Firmicutes and Bacteroidetes.

[0071] It has been found that certain bacteria, such as Firmicutes, have a unique and selective sensitivity to inhibitors of the DNA polIIIC enzyme. Accordingly, 1,7-dihydro-6H-purin-6-one compounds, methods for synthesizing these compounds, and 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, ibezapolstat is 2-((3,4-dichlorobenzyl)amino)-7-(2-morpholinoethyl)-1,7-dihydro-6H-purin-6-one. For instance, ibezapolstat has shown antibacterial activity against a range of Gram-positive bacteria and has been demonstrated to be useful in the treatment of Clostridium difficile infections. Generally, the mechanism of action of DNA pol IIIC inhibitors targets Gram-positive bacteria with a low G+C (G and C DNA bases fewer than A and T bases) content (such as Firmicutes and Bacillales). The DNA polymerase IIIC (DNA polIIIC) enzyme is essential for replication in Gram-positive pathogenic bacteria with a low G+C content (microorganisms whose genomes contain fewer guanine and cytosine bases than adenine and thymine / uracil bases). Thus, LDN analogs that target DNA pol IIIC are selective or can be considered to selectively target Gram-positive pathogenic bacteria with a low G+C genomic 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 with a low G+C genomic content may be more sensitive to LDN analog treatment, while other bacteria (beneficial Firmicutes, Actinobacteria, Bacteroidetes, and / or Gram-negative bacteria) may be insensitive to LDN analog treatment. Although most Gram-positive bacteria require the DNApolIIIC enzyme for replication, the sensitivity of strains to DNApol IIIC inhibitors varies. In addition, differences in sensitivity to small molecule DNA pol IIIC inhibitors (such as LDN analogs) were found among the beneficial Firmicutes strains tested. Surprisingly, not only was there a difference in sensitivity between heterogeneous groups of different species, but also between isolates belonging to the same species (Table 4). As seen in Table 1, a SNP was identified between Y240D (Tyr240Asp) of the sensitive strain Clostridium butyricum 1008 compared to the reference strain of Clostridium butyricum, and two SNPs were identified between Y38D and D146E (Tyr38Asp and Asp146Glu) of the less sensitive strain Clostridium butyricum 1007. These results suggest that small molecule inhibitors of DNA pol IIIC can be designed to target these aspects and enhance selectivity against Gram-positive pathogenic bacteria with a low G+C content. In addition, inhibitors such as LDN analogs can avoid antibiotic resistance.

[0072] The present disclosure can provide methods for improving gut microbiome health. The methods and compositions provided herein can simultaneously reduce the population of physiologically harmful low G+C content Gram-positive pathogenic bacteria in the gut microbiome of an individual while also promoting the persistence, enhanced growth, and / or regrowth of physiologically beneficial gut microbiota.

[0073] In some aspects, the disclosed LDN analogs may have an impact on the flagellar genes (such as fliA, flgB, fliC-VIP) of some bacteria, which may lead to reduced motility of these organisms. The bacteria can be Gram-positive and can include, but are not limited to, Clostridioides difficile.

[0074] Several bacterial species have a single flagellum, and some species have multiple flagella. Although flagella can be considered a motility organelle that enables movement and chemotaxis, flagella may exhibit other functions that vary among bacterial species and change during the bacterial life cycle. In addition, bacterial motility may play an important role in bacterial colonization and survival. For example, flagella allow bacteria to swim and swarm, and thus confer an advantage to such bacterial species in obtaining nutrients, evading predators, and colonizing new environments. In addition, flagella have been shown to be involved in protein export, host cell adhesion, cell invasion, autoagglutination, colonization, regulation of biofilm formation, and may also be associated with the secretion of non-flagellar bacterial proteins involved in virulence processes. Different bacterial species employ different motility mechanisms, including twitching, swimming, gliding, and rotation. In addition to flagella, motility may also involve the use of pili and other appendages, which may be influenced by environmental factors as well as the physical and chemical properties of the surface. For example, some bacterial species can use motility to communicate and form biofilms (Harshey R.M. (2003). Bacterial motility on a surface: many ways to a common goal. Annual review of microbiology, 57, 249-273). Thus, flagella may strongly influence the virulence of bacterial pathogens and may therefore play an important role in bacterial pathogenesis.

[0075] Some examples of Gram-positive bacterial species with at least one flagellum include Listeria monocytogenes, Clostridium botulinum, Clostridium tyrobutyricum, Staphylococcus aureus, Bacillus subtilis, and Clostridium difficile.

[0076] The bacterial flagellum can be located at one position and provide forward movement (polar), or several flagella can be distributed on the bacterial surface (peritrichous) to allow tumbling in place. The flagellum can be a long whip-like appendage that protrudes from the surface of the motile bacterium and rotates to propel the organism. The flagellum consists of three main parts: the filament, which acts as a propeller for bacterial movement and contains the protein flagellin (Flg); the basal body, which contains proteins involved in the rotation of the filament; and the hook, which 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 the flagellin FlgA and attached to the hook protein. The basal body is the motor that drives the rotation of the filament, and it is composed of several proteins (including FliG, FliF, and FliM), and the flagellar basal body rod may include FlgB and FlgC. In addition, the flagellar chaperone protein FlgA may play a key role in early flagellar development (Duan, Q. et al., Flagella and bacterial pathogenicity. Journal of basic microbiology, 53(1), 1-8 (2013).). For example, the regulation of Clostridium difficile motility may occur at least in part at the level of flagellar gene expression.

[0077] In addition to other virulence factors, Clostridium difficile also expresses peritrichous flagella and toxins, which together contribute to the symptoms, pathology, and inflammation of diarrheal diseases. Specifically, the C. difficile flagella can promote bacterial motility and adhesion to intestinal tissues, which in turn aids in gut colonization, a necessary prerequisite for diarrheal disease symptoms. Additionally, it has been observed that the adhesion level of flagellated strains to the murine cecum is 10-fold higher than that 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 proposed that the function and regulation of flagella may contribute to the differences in transmissibility and virulence observed among different C. difficile strains. (Twine, S.M. et al., Motility and flagellar glycosylation in Clostridium difficile. J. Bacteriol. 191:7050-7062 (2009)). Recent studies have also shown that C. difficile, and possibly other Gram-positive anaerobes, may have the ability to alter their virulence factors in a staged manner (i.e., switch between different phenotypes in response to environmental changes) to survive in different environments. Thus, the pathogenicity of C. difficile, including the development of antibiotic resistance, may be one of the consequences of phase variation, and this phase variation may be influenced by flagellar gene expression (Anjuwon-Foster, B.R. & Tamayo, R., Phase variation of Clostridium difficile virulence factors, Gut Microbes, 9:1, 76-83 (2018)).

[0078] Therapeutic administration of the compound

[0079] In some aspects, the LDN analogs provided herein can selectively inhibit the growth of harmful Firmicutes such as Clostridioides difficile. Additionally, the provided compositions and methods can inhibit the growth of the Bacillales order, which includes members of the Staphylococcaceae family, such as Staphylococcus aureus. Thus, the methods and compositions described in this disclosure can promote a healthy and balanced gut microbiome while also reducing or eliminating harmful pathogenic bacteria, which can include MDR strains of low G+C Gram-positive pathogenic bacteria such as C. difficile, S. aureus, drug-resistant S. pneumoniae, and / or Enterococcus faecium. Additionally, the provided LDN analogs can also effectively treat other infections, which can include nosocomial infections. In some aspects, the compositions and methods described herein can reduce the likelihood of or prevent recurrence of infections or dysbiosis caused by low G+C Gram-positive pathogenic bacteria, including C. difficile infections.

[0080] Furthermore, the methods and compositions provided in this disclosure can improve the health of the gut microbiome by administering an effective amount of an LDN analog. The individual does not need to suffer from dysbiosis or infection. The proportion of bacterial phyla in the individual's gut microbiome can be adjusted to a healthier balance compared to the human gut microbiome prior to administration of the LDN analog. For example, upon exposure to the LDN analog, the percentage of Actinobacteria can increase by about 5-50%.

[0081] In some aspects, in patients with C. difficile infection or dysbiosis, the LDN analog causes overgrowth of healthy gut microbiota such as Actinobacteria and Firmicutes species, as well as an increase in the proportion of healthy microbiota such as Clostridiales taxa, during and after treatment. In additional embodiments, in patients with C. difficile infection or dysbiosis, the LDN analog initially increases the abundance of Actinobacteria and then reduces the abundance of Bacteroidetes and increases the abundance of Lachnospiraceae and Ruminococcaceae within 2-3 days after the start of treatment. In yet additional embodiments, the overgrowth of healthy gut microbiota accompanying treatment with the LDN analog results in 100% clinical cure at day 12 and 100% sustained clinical cure at day 38. For example, CDI may be completely eliminated with no recurrence of infection, and the adverse event profile is acceptable.

[0082] In some embodiments, treatment with an LDN analog can attenuate a bacterial infection by targeting one or more virulence factors. In some embodiments, virulence factors can be selectively targeted such that harmful pathogenic bacteria in the gut microbiome are reduced without affecting the healthy microbiota. For example, an LDN analog can inhibit flagellar gene expression in some Gram-positive organisms including, but not limited to, Clostridioides difficile. In some instances, treatment of CDI with an LDN analog can reduce the production of total toxin A and / or B. By targeting bacterial virulence factors, an LDN analog can reduce the overall fitness of bacteria. For example, an LDN analog can modulate bacterial morphology and impair cell division. In some embodiments, an LDN analog can attenuate or eliminate CDI by inhibiting biofilm formation. For example, an LDN analog can reduce the biomass of a biofilm that entraps or contains C. difficile.

[0083] In some aspects, in patients who have not experienced a C. difficile infection or dysbiosis, an LDN analog causes an increase in the abundance of Actinobacteria, mainly Bifidobacteriales or Coriobacteriales, during or after administration. In patients with or without a C. difficile infection or dysbiosis, an LDN analog preserves the proportion of Lachnospiraceae and the abundance of Clostridiales families. In some aspects, an LDN analog improves the health of the gut microbiome by increasing the amount of Clostridium coccoides to improve gut homeostasis. In some additional aspects, an LDN analog can stimulate the immune system to reduce inflammation and allergic diseases and cellular components and metabolites such as butyrate, secondary bile acids, and indolepropionic acid. For example, an LDN analog can support the growth of C. coccoides in the gut microbiome, thereby allowing C. coccoides to produce short-chain fatty acids (SCFAs), which in turn can inhibit the production of pro-inflammatory cytokines.

[0084] Alternatively or additionally, an LDN analog can support the growth of bacterial species that produce anti-inflammatory cytokines. In some additional embodiments, an LDN analog acts as a probiotic, providing energy to enterocytes and strengthening the intestinal barrier. In other aspects, an LDN analog is used prophylactically to prevent, minimize, or reduce dysbiosis.

[0085] An LDN analog can meet the basic criteria of an ideal antibiotic for combating antibiotic-resistant bacterial pathogens that cause many nosocomial infections. For example, the DNA pol IIIC inhibitor ibezaprost achieves high colon concentrations with minimal systemic absorption; has potent activity against C. difficile while causing minimal disruption to the gut microbiome; and is well tolerated in healthy volunteers and CDI patients. Alternatively or additionally, an LDN analog can be used as a prophylactic against dysbiosis.

[0086] According to the method of the present invention, as will be understood by those skilled in the art, the LDN analogs provided herein can be administered to an individual or patient in various forms depending on the selected route of administration. For human or animal use, the LDN analogs can be administered by oral, buccal, rectal, and vaginal routes, or by topical application, and the pharmaceutical compositions are formulated accordingly. Preferably, the LDN analogs are administered in an oral dosage form. Non-limitingly, for oral administration, the composition can be in the form of, for example, tablets, capsules, granules, liquid solutions, and suspensions. The composition can also be administered via suppositories or enemas. For human or animal use, the formulations of the present invention can be administered parenterally, such as intravenously, subcutaneously, intramuscularly, intraorbitally, ophthalmically, intraventricularly, intracranially, intracapsularly, intraspinally, intracisternally, or intraperitoneally, or by intranasal, aerosol, scarification, oral, buccal, rectal, vaginal, or topical application. The formulations of the present invention can also be administered by using a surgical implant that delivers the compound of the present invention in a bolus form or by slow release over a preselected period of time.

[0087] The LDN analogs can be administered to animals, preferably humans, alone or in combination with a pharmaceutically acceptable excipient, in proportions determined by the solubility and chemical nature of the compound, the selected route of administration, and standard pharmaceutical practice, as described above. The LDN analogs can be administered to adults or children. The dosage of the compounds of the present invention and / or the compositions containing the compounds of the present invention can vary depending on many factors, such as the mode of administration; the age, health, and weight of the recipient; the nature and extent of the symptoms; the frequency of treatment and the type of concurrent treatment (if any); and the clearance rate of the compound in the animal to be treated. Those skilled in the art can determine the appropriate dosage based on the above factors. The compounds of the present invention can initially be administered at a suitable dosage, which can be adjusted as needed based on the clinical response. Generally, the compounds of the present invention can be provided as an aqueous physiological buffer solution containing about 0.1 to 10% w / v of the compound or in solid dosage forms such as tablets or capsules. The general dosage range is from about 0.01 mg / kg to about 1 g / kg body weight per day. The oral dosage of ibezaprost can include an amount of about 10 mg to 1000 mg per day, preferably 100 mg to 900 mg per day, and more preferably about 150 mg, 300 mg, 600 mg, or 900 mg per day.

[0088] The LDN analogs can be formulated into pharmaceutical compositions for administration to a human or animal individual in a biocompatible form suitable for in vivo or in vitro administration. Accordingly, the present disclosure provides a pharmaceutical composition comprising a compound of the present invention admixed with an excipient.

[0089] The compounds described herein can be used to treat human intestinal microbial infections caused by Gram-positive bacteria, including strains resistant to common antibiotic drugs. It can also be used to treat related Gram-positive bacterial infections in animals such as pigs, cows, horses, goats, chickens, turkeys, sheep, rats, mice, and rabbits, and to eliminate or avoid bacterial or mycoplasma infections in eukaryotic cell cultures or other media such as food, cosmetics, medical devices, and hospital supplies.

[0090] The compounds of the present invention can optionally be formulated, together with acceptable diluents, carriers, or excipients, and / or in unit dosage forms for pharmaceutical, veterinary, and tissue culture uses. When using the compounds of the present invention, conventional pharmaceutical, veterinary, or culture practices can be employed to provide suitable formulations or compositions, all of which are encompassed in the pharmaceutical compositions of the present invention.

[0091] Non-limitingly, parenteral formulations can be in the form of, for example, liquid solutions or suspensions for oral use, formulations can be in the form of tablets, capsules, liquid solutions, and suspensions (where such solutions and suspensions are particularly suitable for formulations intended for pediatric use); and for intranasal administration, formulations can be in the form of 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 of ordinary skill in the art. Methods well known in the art for preparing formulations can be found, for example, in "Remington's Pharmaceutical Sciences". Formulations for parenteral administration can contain sterile water or saline, ethanol, propylene glycol, polyalkylene glycols (such as polyethylene glycol), oils of vegetable origin, hydrogenated naphthalene, or biocompatible and biodegradable lactide polymers as excipients. 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 can contain lactose as an excipient, or can be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate, and deoxycholate, or can be oily solutions administered in the form of nasal drops, or can be gels for intranasal coating. Formulations for parenteral administration can also include glycocholate for buccal administration, methoxysalicylate for rectal administration, or citric acid for vaginal administration.

[0092] The concentration of the compounds in the formulations of the present invention varies according to many factors, including the dose to be administered and the route of administration. Generally, the compounds of the present invention may be provided in an aqueous physiological buffer solution containing from about 0.1 to 10% w / v of the compound for parenteral administration. The general dose range is from about 0.01 mg / kg to about 1 g / kg body weight per day, for example from about 0.01 mg / kg to 100 mg / kg body weight per day. The dose to be administered depends on the type and extent of the infection to be addressed, the overall health of the patient and the route of administration. For topical and oral administration, the formulations and doses may be similar to those used for other antibiotic drugs.

[0093] In one embodiment, the compounds or compositions of the present invention are administered to an animal, such as a human patient, that has been diagnosed with a Gram-positive bacterial infection. The compounds may also be administered to an animal or a human to inhibit or reduce the likelihood of a Gram-positive bacterial infection, particularly in animals or humans that are prone to such infections (including, but not limited to, immunodeficient or immunosuppressed human patients or those who have recently undergone a medical procedure). In other embodiments, cultured eukaryotic cells are treated with the new compositions, or the compositions are added to inhibit or reduce the likelihood of such infections (e.g., prophylactic treatment). The compounds of the present invention can also be used to prevent bacterial growth in food, cosmetics, and medical supplies, as well as on surfaces.

[0094] The compounds can be administered prophylactically or after an infection has occurred. Prophylaxis may be most appropriate for immunosuppressed human patients and animals, as well as patients and animals after surgery or dental procedures. The list of relevant medical conditions to which the methods of the present invention are applied is not intended to be limiting, and any suitable infection responsive to the compounds can be treated using the methods and / or compounds described herein.

[0095] Reducing the transmissibility of infection

[0096] In some aspects, the LDN analogs provided herein can be used to reduce the transmissibility of Clostridium difficile or other Gram-positive pathogens, for example, by reducing the motility of bacterial cells. In some embodiments, the LDN analogs can inhibit transmission by targeting one or more virulence factors, thereby preventing the spread of harmful diseases. In some embodiments, the LDN analogs can selectively inhibit virulence factors, such as flagellar genes, toxin production, and / or biofilm formation. For example, the LDN analogs can reduce pathogenic transmission by inhibiting flagellar gene expression, thus reducing or eliminating bacterial motility. In addition, the LDN analogs can modulate bacterial morphology and impair cell division. In some embodiments, surface treatment with the LDN analogs reduces or inhibits biofilm formation. In some instances, the LDN analogs can reduce the biomass of biofilms embedded with or containing Clostridium difficile.

[0097] The compound can also be used to treat or coat a culture medium or a surface to prevent microbial growth or reduce the degree of microbial growth. For example, the compounds of the present invention can be mixed with eukaryotic culture media (such as solid or liquid media) to prevent the growth of Gram-positive bacteria. In addition, the compounds of the present invention can be used in surface disinfectant preparations, such as liquid preparations for cleaning and disinfecting surfaces, such as surfaces in kitchens, bathrooms, hospitals or other medical or potential microbial growth areas. 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 medical devices that are wholly or partially implanted into an animal and medical devices outside the animal. Examples of medical devices include, but are not limited to, catheters, dialysis pumps, blood collection devices, stents, and drug delivery devices. Standard preparations for surface treatment or for use in coatings using the compounds of the present invention are known to those skilled in the art.

[0098] Examples

[0099] The foregoing description and examples are set forth only to illustrate the invention and are not meant to be limiting. Since those skilled in the art may envision modifications to the embodiments described in conjunction with the spirit and substance of the invention, the invention should be broadly construed to include all variations within the scope of the claims and their equivalents.

[0100] Example 1:

[0101] Production of DNA polymerase IIIC

[0102] The DNA polymerase IIIC-encoding gene can be amplified from genomic DNA. Primers can be designed such that the 5'-end and 3'-end contain BamHI and XhoI cleavage sites, respectively. The fragment can be directly inserted between the BamHI and XhoI sites of the expression plasmid pET-28a(+). The recombinant protein can have the full-length original protein and have 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 8 primers covering the entire coding region.

[0103] Transfer the plasmid into BL21(DE3) competent Escherichia coli cells. Positive colonies can be inoculated into 1 L of LB medium and incubated with rotation at 200 rpm at 37 °C until an OD600 of 0.6 is reached. Then, 1 mM of the inducer IPTG can be added, and expression can be induced at 16 °C for 18 hours. The 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 protease inhibitor mixture). The cells can be lysed by sonication, and the debris can be centrifuged at 50,000 g for 1 hour.

[0104] The protein can be separated first by passing the lysate through a Ni column. The column can be washed with 50 column volumes (CV) of binding buffer (lysis buffer without protease inhibitor), and then with 10 CV of wash buffer (binding buffer with a total of 40 mM imidazole). The protein can be eluted with elution buffer (binding buffer with a total of 300 mM imidazole).

[0105] Then, the crude extract can be further purified by size exclusion chromatography using a Superdex 200 increase 10 / 300 column with 25 mM Tris-HCl pH 7.5, 0.15 M NaCl, 5% glycerol, and 1 mM DTT. The final product can be stored at -80 °C in 50 - 100 μl aliquots.

[0106] Figure 1 shows the change in the proportional abundance of relevant gut microbiome species over time in healthy volunteers or CDI patients given a DNA pol IIIC inhibitor. Primers targeting Clostridium cluster XIVa (Clostridium coccoides) and Clostridium cluster IV (Clostridium leptum) were used, and qPCR was used to quantify the proportions of these relevant Firmicutes over time. As Figure 1A seen, samples taken from a phase 1 study of healthy volunteers over a 12-day period showed that treatment with 450 mg of a small molecule DNA pol IIIC inhibitor twice daily increased the amount of Clostridium coccoides. Clostridium cluster XIVa, also known as the Clostridium coccoides group, consists of 21 species. These symbiotic bacteria may play an important role in intestinal homeostasis. In addition, members of cluster XIVa have been shown to mitigate inflammatory and allergic diseases, and the cellular components and metabolites of these species, such as butyrate, secondary bile acids, and indolepropionic acid, may act as probiotics in the gut mainly by providing energy to intestinal epithelial cells, strengthening the intestinal barrier, and interacting with the immune system.

[0107] As Figure 1BAs shown, participants with CDI constituted the cohort in the Phase 2a study. In contrast to the healthy volunteers in the Phase 1 study, individuals in Phase 2a showed low levels of Clostridium clusters XIVa and IV throughout the study. However, Tables 2 and 3 show that after initiation of DNA pol IIIC inhibitors (primarily Bifidobacteriales or Coriobacteriales), the abundance of Actinobacteria increased and persisted throughout the dosing period. Compared to the Phase 1 study, the baseline microbiota in the Phase 2a CDI study had a lower proportion of Actinobacteria and Firmicutes and an increased Bacteroidetes. In CDI patients, the abundance of Actinobacteria increased (primarily Coriobacteriales), followed by a decrease in the abundance of Bacteroidetes within 2 - 3 days, and an increase in the abundance of Lachnospiraceae and Ruminococcaceae. Additionally, both the Phase 1 and 2a studies showed that the proportion of Lachnospiraceae was retained, and the abundance of Clostridiales was also retained.

[0108] Determination of minimum inhibitory concentration (MIC) based on broth microdilution

[0109] On the day of testing, the compound can be dissolved in pure DMSO (Sigma 276855 - 2L) to 20 mM as a stock solution. In a V-bottom 96-well plate (Axygen - wipp02280), 30 μl of DMSO can be added to wells 1 to 12 by manual pipetting. 30 μl of the compound DMSO stock solution (20 mM) can be added to well 1 and mixed by pipetting. Two-fold serial dilutions can be performed by transferring 30 μl of the solution from well 1 to well 2 and mixing, then from well 2 to well 3, and so on until well 11. Well 12 can be loaded with 30 μl of DMSO without the compound. This can be the drug "master plate". From wells 1 to 12, the drug concentrations in the master plate can be 10 mM, 5 mM, 2.5 mM, 1.25 mM, 0.625 mM, 0.3125 mM, 0.156 mM, 0.078 mM, 0.039 mM, 0.02 mM, 0.01 mM, and 0 mM in DMSO. Serial dilutions can be performed using a multichannel pipette. Concentrations can be adjusted according to the potency of the compound. A sonic liquid handling system can be used to make sub-plates instead of manual pipetting.

[0110] One day before the MIC test day, the bacterial strain can be streaked from a -80 °C glycerol stock onto an MHA plate and incubated at 37 °C for 20 hours. Streptococcus pneumoniae can be streaked on blood agar and incubated at 37 °C in 5% CO2. A single colony can be picked using an inoculation loop (Greiner - 731175) and suspended in 5 ml of sterile saline. The turbidity of the suspension can be adjusted to 0.10 (Siemens MicroScan turbidimeter), equal to approximately 1.0×10 8cfu / ml. The bacterial suspension can be diluted 100× in the corresponding test medium (Table 1). This can be used to inoculate the sub - plates.

[0111] To prepare the U - bottom 96 - well'sub - plates' (Costar 3788), 98 μl of the test medium was added to each well of the sub - plates. Then aliquots of 2 μl of the solution from the master plate can be replicated and transferred to the sub - plates using a multichannel pipette.

[0112] Aliquots of 100 μl of the bacterial suspension can be inoculated into each well of the sub - plates. Each well contains approximately 5.0×10 5 cfu / ml bacteria, 1% DMSO, and the compound serially diluted in 200 μl of the corresponding test medium, which are 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.56 μM, 0.78 μM, 0.39 μM, 0.2 μM, 0.1 μM, and 0 μM from well 1 to well 12 respectively.

[0113] The plates can be incubated in an environmental atmosphere and a 37 °C incubator for 20 hours.

[0114] The MIC value is determined by visual inspection, which is the lowest compound concentration that completely or significantly inhibits bacterial growth in the test medium.

[0115] The antibacterial activity of the compounds of the present invention against a variety of bacterial organisms was tested, and the bacterial organisms include Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Streptococcus pneumoniae, and Escherichia coli. The Ki of the compounds described in Examples 1, 18, 19, 28, 30, 32, and 34 for the Bacillus subtilis DNA pol IIIC enzyme is 0.31 - 1.45 μM, and the MIC for the Gram - positive organism strains is 0.25 - 4.0 μg / ml. The compounds show weak Gram - negative activity, and the MIC for Escherichia coli is 16 ->64 μg / ml.

[0116] Figure 2 shows the results of MIC assays on selected Firmicutes isolated from samples obtained during the 2a - phase of the study. It was found that among the beneficial Firmicutes strains tested, the sensitivities to DNA pol IIIC inhibitors were different. Surprisingly, not only were the sensitivities different between heterogeneous groups of different species, but also between isolates belonging to the same species (Table 4). The isolated Clostridium butyricum strains (Clostridium butyricum 1008 and Clostridium butyricum 1007) were sequenced and compared. As Figure 2AAs seen, Clostridium butyricum 1008 (1.5 μg / mL) exhibited much lower sensitivity than Clostridium butyricum 1007. In contrast to Clostridium butyricum 1008, Clostridium butyricum 1007 exhibited attenuated sensitivity (Clostridium butyricum 1007 > 100 μg / mL). Figure 2B Figure 2B is a schematic diagram of the molecular structures of two Clostridium butyricum strains. As seen in Table 1 below, compared with the reference strain of Clostridium butyricum, one SNP was identified in Y240D (Tyr240Asp) of the sensitive strain Clostridium butyricum 1008, and two SNPs were identified in Y38D and D146E (Tyr38Asp and Asp146Glu) of the less sensitive strain Clostridium butyricum 1007. These results indicate that small molecule inhibitors of DNApolIIIC have enhanced selectivity for low G+C Gram-positive pathogenic bacteria. In addition, inhibitors provided such as LDN analogs can avoid antibiotic resistance against the targeted pathogenic bacteria.

[0117] Table 1.

[0118]

[0119] In addition, the results indicate that certain beneficial Firmicutes or symbionts exhibit different sensitivities to small molecule inhibitors of DNA pol IIIC. Using isolated gut microbiota species, DNA pol IIIC inhibitors were inactive against representative Actinobacteria (Bifidobacteriaceae and Coriobacteriaceae) and certain Firmicutes (Lachnospiraceae and Lactobacillaceae) (MIC > 64 μg / mL), but highly active against Clostridioides difficile strains (MIC ≤ 2 μg / mL).

[0120] Example 2:

[0121] Microbiome study of a Phase I healthy volunteer study:

[0122] Background: The microbiome of a healthy gut consists of two major groups of bacteria called phyla. Firmicutes (spore-forming Gram-positive organisms) and Bacteroidetes (non-spore-forming Gram-negative organisms) are the most common. The third phylum is Proteobacteria (Gram-negative facultative anaerobes), which is present in low abundance but accounts for 2-5% of the healthy microbiome on average. The fourth phylum is Actinobacteria (mostly Gram-positive bacteria, most of which are saprophytes), which is present in a large proportion in children and the overall proportion usually decreases with age. Patients with Clostridioides difficile infection are in a state of dysbiosis and usually have an increased proportion of Proteobacteria, for example, an excess of Proteobacteria or "Proteobacteria overgrowth", as well as a decrease in the numbers of Firmicutes and Bacteroidetes.

[0123] Ibezaprost Study: Using fecal samples from a Phase 1 healthy volunteer study and shotgun metagenomic sequencing, it was demonstrated that treatment with ibezaprost for 10 days caused a significant difference in the microbiome profile in individuals compared to those receiving placebo. The differences were a greater proportion of Actinobacteria and Firmicutes in individuals treated with ibezaprost, while there was a greater proportion of Proteobacteria in individuals treated with vancomycin.

[0124] Methods and Materials

[0125] Description of the Clinical Trial:

[0126] Six healthy volunteers were given 450 mg of ibezaprost twice daily for 10 days. Fecal samples were collected daily (KW Garey et al., A randomized, double-blind, placebo-controlled, single- and multiple-ascending-dose Phase 1 study to determine the safety, pharmacokinetics, and food and fecal microbiome effects of oral ibezaprost in healthy individuals. Journal of Antimicrobial Chemotherapy 2020;75(12):3635–3643). Approval from the Institutional Review Board was obtained (Midlands Institutional Review Board IRB#222220170383), and all volunteers signed an informed consent form prior to any study procedures. For this analysis, fecal samples were collected daily from days 0 (baseline)–13 and day 30 follow-up from individuals given 450 mg of ibezaprost twice daily, if available. Fecal samples were immediately frozen at -80 °C and then shipped on dry ice to the University of Houston for analysis.

[0127] Fecal DNA Extraction and Shotgun Metagenomic Sequencing:

[0128] As described above, fecal DNA was extracted using the DNAeasy Power Soil Pro kit (Qiagen, catalog number 1288-100) in the QiaCube automated DNA extraction system. (Garey KW, et al. A randomized, double-blind, placebo-controlled, single- and multiple-ascending-dose phase 1 study to determine the safety, pharmacokinetics, and effects of food and fecal microbiome of oral ibezaprost in healthy individuals. Journal of Antimicrobial Chemotherapy 2020;75(12):3635-3643) Shotgun metagenomic sequencing was performed at the Sequencing and Gene Editing Core at the University of Houston (Houston, TX USA), and DNA libraries were prepared using the Nextera DNA Flex library preparation kit and sequenced using the Illumina NextSeq 500 platform. Metagenomic assembly and creation of the abundance table were performed using CLC Genomic Workbench version 12 (Qiagen).

[0129] 16S ribosomal RNA (rRNA) gene sequencing:

[0130] As described in Gonzales-Luna 2021, 16S rRNA sequencing was performed to characterize microbial taxonomy. 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 the gut microbiome community structure. Quality-filtered sequence reads with at least 97% similarity were clustered into operational taxonomic units (OTUs), and species-level taxonomic assignments were made for the representative sequences from each OTU by searching the NCBI 16S rRNA sequence database (release date September 1, 2018) using the NCBI BLAST+ package v2.8.1 2018.

[0131] Shotgun metagenomic sequencing:

[0132] Shotgun metagenomic sequencing was performed on DNA extracted from fecal samples previously used for 16S rRNA sequencing using an Illumina-based platform to analyze microbiome functional genes. Functional gene profiling of shotgun metagenomes was performed using the HUMAnN2 v0.11.2 pipeline.35 In the preprocessing step, sequencing reads were quality filtered and then screened and removed for contaminating host (human) reads. The raw sequence data was filtered and trimmed using Trimmomatic v0.38 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 the nucleotide database (ChocoPhlAn) using bowtie2 and then against the protein database (UniRef90) using diamond. All identified gene families were annotated using UniRef90, and pathways were annotated using MetaCyc identifiers.

[0133] Microbiome analysis

[0134] Using the above sequencing, the proportional abundances of gut microbiome species over time were analyzed in healthy volunteers receiving ibezaprost. The metagenomic data is shown in Table 2 below, which shows the proportion of Firmicutes isolated daily during the study period. The baseline gut microbiota from healthy volunteers was predominantly Firmicutes, Bacteroidetes, or Actinobacteria. Actinobacteria increased in abundance after the start of IBZ (mainly Bifidobacteriales or Coriobacteriales) and persisted throughout the dosing period. As shown in Table 2 below, the genera (species) most frequently isolated during the Phase 1 study in healthy volunteers were Streptococcaceae (Lactococcus lactis and Streptococcus thermophilus); Lachnospiraceae (Blautia, Roseburia); and Ruminococcaceae (Faecalibacterium, Ruminococcus).

[0135] Table 2.

[0136]

[0137]

[0138] Example 3:

[0139] Microbiome data from the Phase 2a clinical trial of ibezaprost for CDI

[0140] Phase 2 clinical trial:

[0141] A Phase 2 clinical trial was designed to evaluate ibezaprost in the treatment of CDI.

[0142] The phase 2a of this trial was an open-label cohort of 10 individuals from U.S. research centers. In this cohort, 10 patients with diarrhea diagnosed by toxin EIA+ as being caused by mild or moderate Clostridioides difficile were treated orally with ibezaprost 450 mg twice daily for 10 days. For recurrence, all patients were followed up for 28±2 days. Stools were collected during the course of the therapy and at follow-up. Clostridioides difficile culture and microbiome changes in the patients' stool samples were evaluated. The study showed that 100% clinical cure was achieved on day 12 and 100% sustained clinical cure was achieved on day 38. Favorable microbiome changes during treatment included overgrowth of Actinobacteria and Firmicutes species. These findings demonstrated that microbiome effects could predict good patient outcomes, including low recurrence rates. The infection was eliminated 100% with no recurrence of infection (100%), and the adverse event profile was acceptable.

[0143] Methods and Materials

[0144] Safety Assessment

[0145] Safety evaluation included AE assessment, physical examination, vital signs, clinical laboratory tests (chemistry, hematology, and urine analysis), and electrocardiogram. Safety endpoints were recorded for all individuals, including the nature, frequency, and severity of AEs. AEs were evaluated at each visit starting from recruitment and were classified according to the Medical Dictionary for Regulatory Activities (MedDRA version 15.0). The severity (mild, moderate, or severe) and causality (not related to the study drug, possibly related, or probably related) of AEs were evaluated by the investigators at each site.

[0146] Microbiology

[0147] To grow Clostridioides difficile, fecal samples were cultured 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 in Texas from 2011 to 2018, including the emergence of ribotype 255. Emerging Microbes & Infections 2020;9(1):341-7). Based on growth and morphology, isolates were identified as Clostridioides difficile, and the Clostridioides difficile toxin and tpi genes were confirmed by PCR. Clostridioides difficile strains were typed using the PCR-based ribotyping method as described previously (Gonzales-Luna AJ, Carlson TJ, Dotson KM, et al. PCR ribotypes of Clostridioides difficile in Texas from 2011 to 2018, including the emergence of ribotype 255. Emerging Microbes & Infections 2020;9(1):341-7). The minimum inhibitory concentration (MIC) of ibezapostat 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)).

[0148] Statistical analysis

[0149] An intention-to-treat analysis was performed on patients who received at least one dose of ibezapolstat. Descriptive statistics for efficacy, safety / tolerance, and PK data were calculated 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). For taxa with a proportional change of at least 1% during the study period, a linear regression model was used to calculate the proportional change of bacterial taxa over the 10-day dosing interval. A p-value < 0.05 was considered significant.

[0150] Results of the Phase 2a clinical trial:

[0151] The completed Phase 2a CDI clinical trial showed a 100% success rate and favorable microbiome changes. Phase 2a data indicated that on the third day of treatment with ibezapolstat, Clostridioides difficile was completely eradicated from the colon, and an overgrowth of healthy gut microbiota, namely Actinobacteriota and Firmicutes species, was observed during and after the therapy. Additionally, the data also showed an increase in the proportion of healthy microbiota, including Clostridiales taxa. The baseline microbiome of the Phase 2a CDI study had a lower proportion of Actinobacteriota and Firmicutes, and an increased Bacteroidota. In CDI patients, the abundance of Actinobacteriota increased (mainly Erysipelotrichales) after the start of IBZ, followed by a decrease in the abundance of Bacteroidota within 2 - 3 days, and an increase in the abundance of Lachnospiraceae and Ruminococcaceae.

[0152] Patients

[0153] Ten patients aged 27 - 75 (±15) years were recruited (50% female; 100% white; 80% Hispanic or Latino). All ten patients received ibezapolstat and completed the study. The median number of unformed stools within 24 hours before starting the therapy was 4 (range: 3 - 10). Two of the ten patients received antibiotics, metronidazole or vancomycin, < 24 hours before starting ibezapolstat. No patient was hospitalized before or after recruitment.

[0154] Microbiological results

[0155] The metagenomic data of the Phase 2a study are shown in Table 3 below, which shows the proportion of Firmicutes isolated daily during the study. In CDI patients, the abundance of Actinobacteria (mainly Coriobacteriales) increased after the start of IBZ, followed by a decrease in the abundance of Bacteroidetes within 2 - 3 days, and an increase in the abundance of Lachnospiraceae and Ruminococcaceae. The most frequently isolated genera (species) during the study included Streptococcaceae (Streptococcus salivarus and Streptococcus thermophilus); Lachnospiraceae (Blautia, Roseburia); Ruminococcaceae (Faecalibacterium, Ruminococcus).

[0156] Table 3

[0157]

[0158] Example 4:

[0159] Gram - positive selectivity profile of ibezaprost

[0160] The aim of this study was to evaluate the selectivity of ibezaprost for the Gram - positive gut microbiota. In vitro and human studies have shown potent activity of ibezaprost against Clostridioides difficile, but selective activity against other beneficial Gram - positive gut microbiota is shown to reduce the risk of CDI recurrence.

[0161] Methods and materials

[0162] Firmicutes fecal isolation

[0163] Using the fecal samples and microbiome data from the above Phase 1 and 2a studies, the change in the proportional abundance of gut microbiome species over time was analyzed in healthy volunteers and CDI patients given ibezaprost. In Phase 1, six healthy volunteers were given 450 mg of ibezaprost twice daily for 10 days. For Phase 1, if available, fecal samples were collected daily from individuals given 450 mg of ibezaprost twice daily at Day 0 (baseline) - Day 13 and Day 30 follow - up. The fecal samples were immediately frozen at - 80 °C and then shipped on dry ice to the University of Houston for analysis.

[0164] The 2a phase of this trial was an open - label cohort of 10 individuals from US research centers. In this cohort, 10 patients with diarrhea diagnosed by toxin EIA+ as caused by mild or moderate Clostridioides difficile were treated orally with 450 mg of ibezaprost twice daily for 10 days. Feces were collected during the course of the therapy and at follow - up.

[0165] Fecal DNA extraction and shotgun metagenomic sequencing:

[0166] As described above, fecal DNA was extracted using the DNAeasy Power Soil Pro kit (Qiagen, catalog number 1288-100) in the QiaCube automated DNA extraction system. (Garey KW, et al. A randomized, double-blind, placebo-controlled, single- and multiple-ascending-dose, phase 1 study to determine the safety, pharmacokinetics, and effects of food and fecal microbiome of oral ibezaprost. Journal of Antimicrobial Chemotherapy 2020;75(12):3635-3643). Shotgun metagenomic sequencing was performed at the Sequencing and Gene Editing Core at the University of Houston (Houston, Texas, USA). DNA libraries were prepared using the Nextera DNA Flex library preparation kit and sequenced using the Illumina NextSeq 500 platform. Metagenomic assembly and creation of the abundance table were performed using CLC Genomic Workbench version 12 (Qiagen).

[0167] 16S ribosomal RNA (rRNA) gene sequencing:

[0168] As described in Gonzales-Luna 2021, 16S rRNA sequencing was performed to characterize microbial taxonomy. 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 the gut microbiome community structure. Quality-filtered sequence reads with at least 97% similarity were clustered into operational taxonomic units (OTUs), and species-level taxonomic assignments were made to the representative sequences from each OTU by searching the NCBI 16S rRNA sequence database (release date September 1, 2018) using the NCBI BLAST+ package v2.8.1

[0169] Shotgun metagenomic sequencing:

[0170] Shotgun metagenomic sequencing was performed on DNA extracted from fecal samples previously used for 16S rRNA sequencing using an Illumina-based platform to analyze microbiome functional genes. Functional gene profiling of shotgun metagenomes was performed using the HUMAnN2 v0.11.2 pipeline.35 In the preprocessing step, sequencing reads were quality filtered and then screened and removed for contaminating host (human) reads. The raw sequence data were filtered and trimmed using Trimmomatic v0.38 with default cutoff settings. Reads were searched against the human genome database using the bowtie2 algorithm in paired-end mode, and if they mapped to the database, they were discarded. To obtain gene family profiles, these quality-controlled metagenomic sequences were first searched against the nucleotide database (ChocoPhlAn) using bowtie2 and then against the protein database (UniRef90) using diamond. All identified gene families were annotated using UniRef90, and pathways were annotated using MetaCyc identifiers.

[0171] Quantitative PCR (qPCR) analysis

[0172] The quantity and quality of the extracted DNA were determined using a Qubit 4 fluorometer (Invitrogen). The sample DNA was diluted to 5 ng / μL with PCR-grade water. Bacterial group DNA levels were evaluated using specific PCR primers / conditions. 11-14 Using a 7300 Real-Time PCR System (Applied Biosystems), qPCR was performed in triplicate for each sample in a final volume of 20 μL containing 25 ng of DNA template, 0.5 μM of primer, and QuantiTect SYBR Green Mix (Qiagen). For eubacteria, a 0.25 μM FAM-labeled probe and TaqPath ProAmp Premix (Qiagen) were used. Threshold cycle values were converted to copies per ng of DNA using a standard curve. Standards were prepared by performing PCR on appropriate bacterial strains or DNA from normal feces using species-specific primers. PCR products were cloned using the Invitrogen TOPO PCR Cloning Kit (Invitrogen) and verified by sequencing at the University of Houston Core Facility. A Basic Local Alignment Search Tool (BLAST) search was performed to identify the most closely matching database sequences. A series of 10-fold serial dilutions of plasmid standard DNA (5 × 10 8 to 500 copies) were run in triplicate on each qPCR plate. The standard curve R of the standards was calculated 2Values. Considering the initial sample DNA concentration and fecal weight, the copy number per gram of feces was calculated. The change in bacterial levels (Δlog10 copies / g of feces) for each participant from the entry level to each available consecutive time point was determined, and the median change was calculated.

[0173] Firmicutes fecal isolates (phase 2a samples):

[0174] 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 each of glucose, maltose, and cellobiose in large (13.5 cm diameter) petri dishes. The full-length 16S rRNA gene was amplified by PCR (using the 7F (5'-AGAGTTTGATYMTGGCTCAG-3') forward primer and 1510R

[0175] (5'-ACGGYTACCTTGTTACGACTT-3') reverse primer), and then Sanger sequencing was performed to identify the isolated colonies. Using the Ribosomal Database Project (RDP) as a reference, the full-length 16S rRNA gene sequence reads were aligned using CLC Genomics (Qiagen) to classify the reads into operational taxonomic units (OTUs). The full-length 16S rRNA gene sequence of each species-level OTU was compared with the RDP reference database to assign taxonomic names to the genus level, and a BLAST search was performed to identify any candidate novel species.

[0176] Whole-genome sequencing

[0177] Genomic DNA was extracted from at least one representative of each identified species. The DNA was sequenced on the Illumina HiSeq platform, generating 150 bp read lengths, and these were assembled and annotated for further analysis. For whole-genome SNP analysis, using the developers' guidelines (https: / / github.com / katholt / RedDog), the cleaned sequence reads were mapped to the reference genome using CLC Genomics and the RedDog pipeline. Briefly, Bowtie2 version 2.2.3 was used for mapping, and SAMtools version 0.1.19 was used for calling SNPs. Only high-quality SNPs were used for phylogenetic analysis.

[0178] Minimum inhibitory concentration (MIC) determination:

[0179] The strain was streaked onto blood agar (Hardy Diagnostics) and incubated at 37 °C for 24 h in an anaerobic environment (Coy ethylene anaerobic chamber). After incubation, 3-5 well-isolated colonies were suspended in 5 mL of BHI broth (standard medium). The culture was incubated in the anaerobic chamber for 24 h to reach a 0.5 McFarland Standard. Brucella agar had 5% hemin (5 μg / mL) (Sigma), vitamin K (10 μg / mL), and defibrinated sheep blood (Northeast Lab Services). The supplemented Brucella agar was then used for two-fold serial dilutions to produce plates with IBZ concentrations of 0.5 to 16 μg / mL per plate. The plates were allowed to dry in a sterile location. After drying, the plates were covered with foil to prevent any light damage to the hemin and placed in the anaerobic chamber for 1 h to condition the medium. The broth culture was then spotted onto the plates and covered with foil. The plates were allowed to dry completely and then incubated upside down for 48 h. The plates were analyzed and the MIC was recorded.

[0180] Microbiological results:

[0181] The baseline gut microbiota from healthy volunteers was predominantly Firmicutes, Bacteroidetes, or Actinobacteria. The abundance of Actinobacteria increased (mainly Bifidobacteriales or Coriobacteriales) after the start of ibezapostat and persisted throughout the dosing period. In the Phase 1 and 2a studies, the proportion of Lachnospiraceae and the abundance of Clostridiales families were both preserved. Compared to the Phase 1 study, the baseline microbiota in the 2a CDI study had a lower proportion of Actinobacteria and Firmicutes and an increased Bacteroidetes. In CDI patients, the abundance of Actinobacteria increased (mainly Coriobacteriales) after the start of ibezapostat, followed by a decrease in the abundance of Bacteroidetes within 2-3 days, and an increase in the abundance of Lachnospiraceae and Ruminococcaceae.

[0182] Beneficial Firmicutes showed mixed sensitivity to ibezapostat

[0183] Using isolated gut microbiota species, ibezapostat was inactive against representative Actinobacteria (Bifidobacteriaceae and Coriobacteriaceae) and certain Firmicutes (Lachnospiraceae and Lactobacillaceae) (MIC > 64 μg / mL), but highly active against Clostridioides difficile strains (MIC < 2 μg / mL). Thus, the microbiome changes induced by ibezapostat may depend on the underlying composition of the baseline microbiome. However, in the Phase 1 and 2a cohorts, the abundance of Actinobacteria increased after the start of therapy. The combination of IBZ microbiome data with in vitro MIC assays demonstrated that the persistence or regrowth of a healthy microbiome is associated with beneficial physiological effects. The MIC assay results for Firmicutes are shown in Table 4. Among the isolated beneficial Firmicutes, ibezapostat showed mixed sensitivity.

[0184] Table 4.

[0185]

[0186]

[0187] Although Firmicutes generally possess the DNA Pol IIIC enzyme, some members of the Firmicutes (also known as Bacillota) are more sensitive to ibezapostat. Among the heterogeneous group of Firmicutes tested, different strains of Clostridium butyricum showed different sensitivities. As Figure 1A seen, Clostridium butyricum 1008 was more sensitive to ibezapostat (IBZ MIC: 1.5 μg / mL) compared to Clostridium butyricum 1007 (IBZ MIC: > 100 μg / mL). After whole-genome sequencing as described above, three single nucleotide polymorphisms (SNPs) were identified. As seen in Table 1, one SNP was identified in Y240D (Tyr240Asp) of the sensitive strain Clostridium butyricum 1008, and two SNPs were identified in Y38D and D146E (Tyr38Asp and Asp146Glu) of the less sensitive strain Clostridium butyricum 1007, compared to the reference strain of Clostridium butyricum. The results suggest that targeted ibezapostat drug development against Clostridioides difficile may result in intermittent activity against beneficial symbionts.

[0188] Example 5:

[0189] Novel pharmacology and sensitivity of ibezapostat against Clostridioides difficile isolates with reduced sensitivity to antibiotics directed against Clostridioides difficile

[0190] The aim of this study was to evaluate the efficacy of ibezapostat against Clostridioides difficile strains showing reduced sensitivity to current CDI antibiotics. Additionally, this study also evaluated the role of motility inhibition and IBZ treatment in flagellar gene expression in Clostridioides difficile strains with reduced sensitivity to current CDI antibiotics.

[0191] Methods and materials

[0192] The goal of this study was to evaluate the sensitivity of strains with reduced susceptibility to current CDI antibiotics to IBZ and to evaluate motility inhibition.

[0193] Isolates:

[0194] Twelve clinical isolates with reduced susceptibility to metronidazole (MIC range: 0.25 - 8 μg / mL), vancomycin (MIC range: 1 - 16 μg / mL), or fidaxomicin (<0.03125 - 2 μg / mL) were tested.

[0195] Minimum inhibitory concentration (MIC) determination:

[0196] An agar dilution MIC study was performed on Clostridioides difficile strains with reduced susceptibility to metronidazole, vancomycin, and fidaxomicin according to the guidelines for anaerobes in the CLSI document M11 - A7. Cultures of Clostridioides difficile were prepared by inoculating a bacterial colony into BHI supplemented with 0.1% sodium taurocholate. After incubation for 24 hours at 37°C in an anaerobic chamber, the pre - culture was diluted 1:100 in fresh BHI containing the appropriate concentration of antibiotic to approximately 10 6 CFU / mL. Antibiotic concentrations were prepared by a two - fold dilution series (1 concentration per agar plate) according to CLSI guidelines. For example, for each of the three antibiotics, namely vancomycin, ibezapolstat, or metronidazole, the antibiotic concentration ranged from approximately 64 μg / ml to approximately 0.25 μg / ml, and for fidaxomicin, the concentration ranged from approximately 16 μg / ml to approximately 0.03 μg / ml.

[0197] At 24 hours, the inhibitory concentration was determined by visual inspection. As shown in Table 5, the MIC values were correlated with the susceptibility to the antibiotic with reduced susceptibility (difference >8×MIC value), where the MIC was expressed in mg / L. The results showed that IBZ maintained its efficacy against clinical isolates with reduced susceptibility to metronidazole, vancomycin, and fidaxomicin. In addition, there was no difference in the IBZ MIC50 and MIC90 between susceptible isolates and isolates with reduced susceptibility.

[0198] Table 5. IIBZ maintains activity against resistant strains

[0199] Metronidazole Vancomycin Fidaxomicin Ibezaprost MT 4802 0.25 1 0.006 4 MT 5529 2 1 0.5 4 SH 1132 1 4 0.5 8 MT 5342 2 8 1 4 MT 5364 2 4 1 4 MT 5426 4 4 0.5 8 MT 5515 4 4 2 8 MT 4883 2 8 2 8 MT 5493 2 1 1 4 MT 5536 0.25 2 1 8 MT 5382 2 1 1 8 MT 5071 1 4 1 8

[0200] Quantitative PCR (qPCR) analysis

[0201] The motility of Clostridioides difficile was evaluated using quantitative RT-PCR. After pretreatment with sub-MIC concentrations of IBZ by adapting the method of Doan et al. (Antibiotics 2022), the expression of relevant flagellar genes (fliA, flgB, fliC-VIP) in the reference Clostridioides difficile strain CD 630 was quantified by qPCR. Briefly, the bacterial pre-culture was diluted and then grown at 37 °C under anaerobic conditions in BHI with sub-inhibitory concentrations of IBZ (0.5×MIC) for 4 hours. The transcript levels of fliA, flgB, and fliC were measured and compared with the control gluD recombinant protein (Clostridioides difficile).

[0202] Figure 3 The target genes are shown on the x-axis, and the relative expression of these genes compared to the untreated control is shown on the y-axis. Additionally, to visualize the data more vividly, the relative expression of the control was normalized to a value of 1. Thus, Figure 3 it was shown that when treated with IBZ, the expression of fliA was attenuated by approximately 60%, the expression of flgB was reduced by approximately 30%, and the expression of fliC was reduced by approximately 80% compared to the untreated control. Thus, qPCR analysis indicated an overall 2- to 5-fold reduction in flagellar gene expression after sub-MIC IBZ exposure.

[0203] Motility assay:

[0204] The motility of Clostridioides difficile was evaluated using a phenotypic motility assay. Briefly, a culture of CD 630 was prepared by inoculating BHI medium containing 0.3% agar and sub-inhibitory concentrations of IBZ and grown anaerobically at 37 °C for 48 hours. After incubation, the bacterial strain was placed in semi-solid BHI agar for 4 hours and motility was visually observed.

[0205] Figure 4 The results of the motility assay are shown. As Figure 4 seen, the control strain was able to swim unrestrained, and thus it could be seen to migrate freely from the inoculation vertical line. On the other hand, the strain exposed to sub-inhibitory (non-killing) concentrations of IBZ in the medium was seen to remain near its inoculation point. Such results indicate that the motility of the culture of CD 630 was lower due to the presence of IBZ in the medium.

[0206] The results showed that IBZ retained activity against Clostridioides difficile strains with reduced susceptibility to other commonly used antibiotics and exhibited new pharmacological properties, which may be due to its unique mechanism of action.

[0207] Example 6:

[0208] Metagenomic evaluation of ibezapostat compared with other anti-Clostridioides difficile agents

[0209] Published studies have shown that in humans, treatment with ibezaprost (IBZ) may cause less disruption of the microbiome than treatment with vancomycin (see, for example, Figure 5 ). Nevertheless, no comparative microbiome studies have been conducted for other anti - Clostridioides difficile antibiotics. The aim of this study was to compare the in - vivo changes in the gut microbiome in response to treatment with IBZ and other anti - Clostridioides difficile antibiotics. Thus, this study aimed to compare the perturbation of the gut microbiome from IBZ to three other anti - Clostridioides difficile antibiotics, including vancomycin (VAN), fidaxomicin (FDX), or metronidazole (MTZ).

[0210] Methods and Materials

[0211] Germ - free (GF) mice (six per group) were randomly assigned to IBZ, VAN, FDX, MTZ, or a control group. Figure 6 The basic experimental design of the humanized germ - free mouse study is shown.

[0212] Germ - free (GF) humanized mice

[0213] Healthy human - sourced fecal slurry was orally gavage - fed to GF mice. The humanized mice were housed in a germ - free biological safety cabinet (BSC) for one week to allow the establishment of the donor gut microbiome. Then the humanized mice were moved to germ - free micro - isolator cages and the pellet diet was changed to a powder diet. Then the humanized mice were acclimated to the powder diet for one week. In addition, fecal samples were obtained before the diet change (baseline 1). On day 14, once the mice were acclimated to the powder diet, a second baseline fecal sample (baseline 2) was obtained and antibiotic treatment was initiated. The appropriate antibiotic was added to the powder diet for 10 days. On day 16, two days after the start of antibiotic treatment, another fecal sample (ABX - 1) was collected. During the 10 - day antibiotic treatment period, the powder diet trays were replenished daily with fresh feed containing the randomly assigned antibiotic. Fecal samples were collected again on day 24 (ABX - 2).

[0214] Microbiome analysis

[0215] To assess the microbiome impact of the antibiotics, 16S rRNA metagenomics was used on fecal samples collected on day 0 (baseline 1 and baseline 2) and 2 and 14 days after antibiotic treatment. Using The kit extracts a large amount of DNA from fecal samples. The V4 region of the bacterial 16S rRNA gene was sequenced using the Illumina MiSeq and CLC Genomics Workbench. The raw sequencing reads were processed and curated using the mothur (v.1.48.0) software package and followed the mothur MiSeq SOP outlined in: 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): pp. 7537-41 (see also 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): pp. 5112-20.). All data visualization and statistical analyses were performed using R studio (R Core Team (2023). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria).

[0216] Results

[0217] Before antibiotic initiation, the Shannon index alpha diversity was similar between treatment groups.

[0218] Microbiome analysis

[0219] Figure 7A -E presents boxplots with lines connecting groups based on color, where the color indicates the antibiotic to which the mice were exposed during the last 10 days of the experiment (or the lack of antibiotic in the no-drug control (ND control) and baseline samples). Figure 7A and7B showed the changes in α-diversity of the gut microbiome throughout the experiment, and these figures showed the changes in the richness of operational taxonomic units (OTUs) within the gut microbiome during the experiment ( Figure 7A ) and the changes in the inverse Simpson index within the gut microbiome during the experiment ( Figure 7B ). For both metrics, dietary changes followed by exposure to different combinations of antibiotics significantly affected the inverse Simpson index (ANOVA, p < 0.001). As Figure 7A and 7B showed, α-diversity was significantly reduced in all antibiotic groups compared to the control (p < 0.05).

[0220] Figure 7C , 7D and 7E showed the changes in β-diversity (diversity between groups) of the gut microbiome throughout the experiment and showed different clustering in mice given IBZ compared to all other antibiotics. Figure 7C showed the changes in β-dispersion (distance to the centroid) of the gut microbiome throughout the experiment, Figure 7D showed the changes in Bray-Curtis dissimilarity from baseline 1 (day 7) throughout the experiment, and Figure 7E showed the changes in Bray-Curtis dissimilarity from baseline 2 (day 14) throughout the experiment. For all metrics of β-diversity, dietary changes followed by exposure to antibiotics significantly increased the Bray-Curtis dissimilarity from baseline 2 (day 14) (ANOVA, p < 0.001 for all).

[0221] Figure 7F provided non-metric multidimensional scaling analysis (NMDS) of Bray-Curtis dissimilarity.

[0222] Figure 7A - 7EThe box plots presented include points representing one sample taken at the corresponding time point (marked at the top of each plot), where the size indicates the inverse Simpson index value of the sample, the shape indicates from which trial the sample was collected, and the color indicates to which antibiotic (or lack thereof) each sample was exposed. When comparing the exposed groups (colors) at two time points (ABX-1 and ABX-2) in the group exposed to antibiotics, there was a statistical difference between the antibiotic-exposed groups (PERMANOVA; for both time points, p < 0.001). Additionally, when comparing Trial 1 and Trial 2, there were statistical differences between the two trials at all time points (Baseline 1, Baseline 2, ABX-1, and ABX-2) (PERMANOVA; p < 0.001). Table 6 below depicts the changes in the diversity metrics within the gut microbiome observed in the treated humanized mice and indicates comparisons between antibiotic treatments. For each antibiotic (ibezaprost, fidaxomicin, vancomycin, and metronidazole), comparisons were made to Baseline 2 (Day 14). The comparisons at Day 7 and Day 14 were made using Baseline 1 (Day 7) as a reference point to account for diversity changes due to diet changes. Significance codes were determined based on the following p-value parameters: approximately 0 = "***", approximately 0.001 = "**", approximately 0.01 = "*", approximately 0.05 = ".". Any case with a p-value > 0.05 was not considered significant.

[0223] Table 6.

[0224]

[0225]

[0226] Figure 8A - 8D Stacked bar charts are provided that illustrate the average relative abundances (expressed as percentages) of different bacterial taxonomic ranks throughout the experiment. Thus, 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; furthermore, Table 7 below depicts the changes in the relative abundances of bacterial phyla within the gut microbiome observed in the treated humanized mice and indicates comparisons between antibiotic treatments. For each antibiotic (ibezaprost, fidaxomicin, vancomycin, and metronidazole), comparisons were made to Baseline 2 (Day 14). The comparisons at Day 7 and Day 14 were made using Baseline 1 (Day 7) as a reference point to account for diversity changes due to diet changes. Significance codes were determined based on the following p-value parameters: approximately 0 = "***", approximately 0.001 = "**", approximately 0.01 = "*", approximately 0.05 = ".". Any case with a p-value > 0.05 was not considered significant.

[0227] Table 7

[0228]

[0229]

[0230] Figure 9A - 9E Bar plots of OTUs identified by random forest analysis that distinguish the specified treatment groups are provided. The dashed line indicates the significance cut-off based on the median importance level, assuming 1 standard deviation in both directions, and the OTUs shown (x-axis) are classified to the family level. Additionally, for each group, the OTUs that are compared and enriched (elevated) as shown in the legend are colored. Figure 9A -D depicts the comparison of each antibiotic-exposed mouse to the drug-free control group (ND control), and Figure 9E compares mice exposed to ibezapolstat to mice exposed to fidaxomicin.

[0231] At the phylum level, a significant increase in the proportion of Bacteroidetes was observed in the IBZ group, while an increase in Firmicutes was observed in the FDX group. Additionally, the relative abundances of Proteobacteria and Verrucomicrobia increased in the VAN and MTZ groups.

[0232] Conclusions

[0233] These studies show that the change in food from pelleted to powdered feed reduces α-diversity, causes a slight decrease in the abundance of Bacteroidetes, and an increase in Verrucomicrobia (Akkermansia). Although all antibiotics disrupt the gut microbiome, the changes appear to be drug-dependent. Generally, ibezapolstat and fidaxomicin cause an increase in the proportion of Bacteroidetes, while vancomycin and metronidazole cause an increase in the proportion of Proteobacteria.

[0234] Furthermore, compared to the well-known gut microbiota disruptors vancomycin and metronidazole, administration of IBZ and fidaxomicin to human microbiota GF mice both caused favorable but distinct changes in the gut microbiome. These results support the continued clinical development of IBZ for the treatment of CDI.

[0235] Example 7:

[0236] Ibezapolstat Regulates Clostridioides difficile Virulence Factors In Vitro

[0237] In the phase I and IIa clinical trials conducted to date, the use of ibezapostat has shown favorable effects on the gut microbiome, which can predict anti-relapse pharmacological properties. Therefore, the aim of this study was to evaluate the effect of IBZ on Clostridium difficile virulence factors. To facilitate this aim, the study investigated the effects of sub-inhibitory concentrations of IBZ on the cell morphology, motility, and toxin production of C. difficile in vitro. Based on its potentially novel mechanism of action targeting the DNA pol IIIC enzyme, IBZ may exhibit unique pharmacological properties beyond killing bacteria.

[0238] Methods and Materials

[0239] Clostridium difficile reference strains CD 630 and R20291 were treated with ibezapostat at sub-inhibitory concentrations up to the minimum inhibitory concentration. Concentrations of toxin A and toxin B were measured by ELISA (tgcBiomics). After treatment with IBZ for 4 hours, the expression of flagellar genes fliA, flgB, and fliC in CD 630 was evaluated using RTqPCR. Morphological changes induced by treatment with IBZ were evaluated by bright-field microscopy at 10 - 40x magnification.

[0240] Morphological Evaluation

[0241] Cultures were prepared by inoculating a C. difficile colony into BrainHeart 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 pre-cultures were diluted to approximately 10 6 CFU / mL (1:100) in fresh BHI containing the appropriate antibiotic at 0.25×MIC (0.5 μg / mL). After incubation for 24 hours, bright-field photographs were taken using an EVOS imaging system (Thermofisher) at 40x magnification.

[0242] Motility Assay

[0243] Phenotypic motility assays were used to evaluate C. difficile motility. Cultures of bacterial strains were prepared by inoculating BHI medium containing 0.3% agar and sub-inhibitory concentrations of IBZ and grown anaerobically at 37 °C for 48 hours. After incubation, the bacterial strains were placed in semi-solid BHI agar for 76 hours and motility was visually observed.

[0244] Expression of Flagellar Genes

[0245] Dilute the bacterial pre - culture in BHI and grow it for 4 hours at 37 °C under anaerobic conditions in the presence of sub - inhibitory concentrations of antibiotics. To assess flagellar gene expression, measure the transcript levels of fliA, flgB, and fliC by qRT - PCR (Doan et al. 2022).

[0246] Toxin production

[0247] Dilute the bacterial pre - culture in BHI and then grow it for 24 hours in the presence of sub - inhibitory concentrations of antibiotics. Assess toxin production by ELISA (tgcBiomics) according to the manufacturer's instructions.

[0248] Results

[0249] In general, ibezaprost reduces the concentrations of toxin A and toxin B in a dose - dependent manner. After normalizing toxin production relative to the control, a 55% (CD 630) to 60% (R20291) reduction in toxin levels was observed. It was also observed that motility and flagellar genes were reduced by up to 50% after treatment with 0.25×MIC ibezaprost for 4 hours. And finally, both the CD 630 and R20291 strains exhibited an elongated cell phenotype at sub - inhibitory to MIC levels of ibezaprost.

[0250] Morphological assessment

[0251] Figure 10 Shows the effect of ibezaprost on the morphology of CD 630. As Figure 10 shown, ibezaprost shows a dose - dependent effect on the cell length of Clostridioides difficile, and it affects the cell division pathway of Clostridioides difficile after 24 hours of treatment. This phenotype has been observed previously with other DNA - targeting antibiotics.

[0252] Motility assay

[0253] Figure 11 Shows the effect of ibezaprost on the motility of CD 630. As Figure 11 seen, treatment of CD 630 with sub - inhibitory concentrations of ibezaprost in semi - solid BHI agar for at least 48 hours (up to 76 hours) results in reduced motility.

[0254] Expression of flagellar genes

[0255] Figure 12 Shows the effect of ibezaprost on flagellar gene expression. After treatment with sub - inhibitory concentrations of IBZ (0.5×MIC) for 4 hours, CD 630 shows a 2 - to 5 - fold reduction in flagellar genes compared to the gluD control ( Figure 12 ).

[0256] Toxin production

[0257] Figure 13 showed the effect of ibezapostats on modulating Clostridium difficile toxin production. As Figure 13 shown, IBZ decreased the production of total toxins A and B in strain CD 630 in a dose-dependent manner after 24 h of treatment.

[0258] Conclusion

[0259] As Figure 10 - 13 shown, ibezapostats treatment exhibited in vitro effects on the virulence determinants of Clostridium difficile. At sub-inhibitory levels, ibezapostats affected several aspects of Clostridium difficile virulence. For example, IBZ affected bacterial fitness by modulating morphological cell division damage. As visually observed in the motility assay seen in Figure 11 , IBZ also decreased Clostridium difficile motility. And further, IBZ attenuated the relative expression of the flagellar genes fliA, flgB, and fliC, and decreased the production of toxin A and toxin B of Clostridium difficile. These preliminary results elucidated the mechanism of action of IBZ and supported its continued clinical development.

[0260] Example 8:

[0261] In vitro biofilm study of ibezapostats and comparator antibiotics against Clostridium difficile

[0262] It was hypothesized that the high recurrence rate of Clostridium difficile infection (CDI) was partly attributed to biofilm formation. Although IBZ has been shown to be a novel pol IIIC DNA polymerase inhibitor antibiotic and had a low CDI recurrence rate in phase II clinical trials, its in vitro effect on Clostridium difficile biofilm was unknown. Therefore, the aim of this study was to compare the anti-biofilm activities of IBZ with comparator antibiotics against Clostridium difficile.

[0263] Methods and materials

[0264] To observe the effects of comparing IBZ and comparator antibiotics on Clostridium difficile biofilm, antimicrobial activity and biomass studies of biofilms were used. In these studies, IBZ was compared with the antimicrobials vancomycin (VAN), fidaxomicin (FDX), and metronidazole (MTZ). The basic flow chart outlining the experimental procedures used in these studies can be seen in Figure 14 .

[0265] Biofilm formation

[0266] Briefly, the Clostridium difficile laboratory strain R20291 was grown in a medium supplemented with Brain Heart Infusion (BHIS). To initiate biofilm cultivation, 100 μL of the Clostridium difficile culture (OD600nm = 0.01) was added to each well of a 96-well plate. Biofilm formation was established by incubating the cultures anaerobically at 37 °C for 24 hours. Early biofilm formation was established at 4 hours, while late biofilm formation was established at 24 hours, 48 hours, and 72 hours.

[0267] Antimicrobial activity

[0268] After biofilm formation, the BHIS medium was removed and replaced with fresh medium without antibiotics (control), ibezapostat (IBZ), fidaxomicin (FDX), or metronidazole (MTZ). The cultures were then incubated anaerobically for 24 hours or for time-course testing.

[0269] Biofilm biomass

[0270] Biofilm biomass was measured using crystal violet staining. The 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. The data were analyzed by comparing the growth percentage of antibiotic-treated cultures to the control.

[0271] Results

[0272] Figure 15 Early (24 hours) and late (>72 hours) biofilm formation of biofilms embedded with the R20291 or CD 630 Clostridium difficile strains are shown. Figure 17 Panels A and 17B show that IBZ and vancomycin (VAN) inhibit Clostridium difficile biofilm formation and reduce Clostridium difficile biofilm biomass at multiple time points. Compared to the control, IBZ and VAN reduce Clostridium difficile growth (CFU / mL) and biomass at sub-MIC (0.4X MIC), and in early biofilms, supra-MIC (40X MIC) VAN reduces growth, while IBZ eradicates growth after 48 hours of treatment (see Figure 17 Panels A and 17B).

[0273] Antimicrobial activity

[0274] Figure 16 Twenty-four-hour biofilms embedded with R20291 or CD 630 Clostridium difficile are shown. As Figure 16 seen, the minimum bactericidal concentration is similar to the vegetative non-biofilm growth for all four antibiotics, although the Eagle effect of MTZ is observed at higher concentrations.

[0275] Biofilm biomass

[0276] As mentioned above, Figure 17 A and 17B show the effects of IBZ and VAN on early biofilms (i.e., biofilms formed within 4 hours). As Figure 17 seen in A, measured as CFU / mL, both IBZ and VAN reduced biofilm growth within 48 hours at MIC and sub-MIC levels. Additionally, Figure 18 showed that late biofilms (i.e., biofilms formed within >24 hours) embedded with R20291 or CD 630 exhibited biphasic growth, with a reduction in biomass at 48 hours and 24 hours, and regrowth observed at 72 hours (possibly due to biofilm detachment). As Figure 18 shown, although differences were noted, overall all antibiotics reduced biofilm biomass regardless of biofilm growth time compared to the control, and the highest reductions were observed during later biofilm growth periods (48 hours and 72 hours). Additionally, Table 8 provides the MIC and MBIC of the test antibiotics applicable to Clostridium difficile strain R20291 in μg / mL.

[0277] Table 8

[0278] Compound MIC (μg / mL) MBIC (μg / mL) Vancomycin 1 1 Ibezaprost 4 4 Fidaxomicin 0.06 0.125 Metronidazole 0.5 0.5

[0279] Conclusion

[0280] These studies suggest that IBZ may be as effective as comparable antibiotics in reducing the amount of Clostridium difficile embedded in biofilms and biofilm biomass. These results, together with the success of clinical trials to date, warrant the continued development of IBZ.

[0281] Other Embodiments

[0282] Those skilled in the art will recognize or be able to determine many equivalents of the specific procedures described herein using only routine experimentation. All such equivalents are considered to be within the scope of the present invention. Various substitutions, alterations, and modifications can be made to the present invention without departing from the spirit and scope thereof. Other aspects, advantages, and modifications are within the scope of the present invention. The contents of all references, issued patents, and published patent applications cited throughout this application are incorporated herein by reference. Suitable components, processes, and methods of those patents, applications, and other documents can be selected for the present invention and its embodiments.

[0283] References

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[0327] R Core Team (2023). R: A language and environment for statistical computing, Vienna, Austria).

Claims

1. A method for promoting the health of an individual's gut microbiome, comprising: administering to an individual suffering from gut microbiota dysbiosis an effective amount of a low G+C directed nucleoside (LDN) analogue; wherein the LDN analogue simultaneously reduces harmful Gram-positive bacteria in the gut microbiome having genomes with low guanine and cytosine (low G+C) content while maintaining and / or increasing beneficial microorganisms in the gut microbiome.

2. The method according to claim 1, wherein the LDN analogue is a small molecule inhibitor of DNApol IIIC enzyme.

3. The method according to claim 2, wherein the LDN analogue targets DNApol IIIC of low G+C bacteria having genomes containing <50% guanine (G) + cytosine (C).

4. The method according to 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. The method according to claim 1, wherein the beneficial microorganisms comprise Firmicutes and include Lachnospiraceae and Lactobacillaceae.

6. The method according to claim 5, wherein administering the effective amount of the LDN analogue reduces the growth or prevents the regrowth of low G+C content Gram-positive pathobionts in the gut microbiome within 30 days.

7. A method for achieving and / or maintaining a healthy proportion of gut microbial communities in an individual's intestinal environment, comprising: administering to the individual an effective amount of a compound targeting DNA pol IIIC enzyme in Gram-positive pathobionts with low G+C content; reducing or eliminating physiologically harmful pathogenic microorganisms belonging to the Bacillota phylum; and increasing and / or maintaining physiologically beneficial microorganisms in the intestinal environment.

8. The method according to claim 7, wherein the compound is a low G+C directed nucleoside (LDN) analogue.

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

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

11. The method according to claim 8, wherein administering the LDN analogue is prophylactic, the individual is healthy, and the LDN analogue restores or maintains a mutualistic relationship between the individual and the microorganisms in the intestinal environment.

12. The method according to claim 7, wherein the proportion of bacterial phyla in the gut microbiome of the individual is adjusted to a healthier balance compared to the gut microbiome of the individual prior to administering the LDN analogue.

13. The method according to claim 8, wherein the physiologically beneficial microorganisms in the intestinal environment comprise anaerobic Gram-positive bacteria belonging to the genus Clostridium and include Clostridium coccoides.

14. The method according to claim 12, wherein the individual's gut is suffering from an overgrowth of Clostridioides difficile.

15. The method according to claim 11, wherein the LDN analogue is continuously administered until most of the bacterial species are from the phylum Actinobacteria, Firmicutes or Bacteroidetes and a small portion are from the phylum Proteobacteria.

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

17. The composition according to claim 16, wherein the LDN analogue is a prophylactic therapeutic agent and promotes the persistence and / or regrowth of a healthy microbiota.

18. The composition according to claim 16, wherein the individual receives at least 450 mg of the LDN analogue at least once a day.

19. The composition according to claim 16, wherein the LDN analogue selectively reduces the growth of pathogenic members of Streptococcus, Enterococcus, Staphylococcus, Bacillus, Clostridium, Streptococcus pneumoniae, Listeria, Mycoplasma and / or Lactobacillus.

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

21. The composition according to claim 20, wherein the LDN analogue inhibits the motility of the Gram-positive organisms by reducing flagellar gene expression.

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