Using Ibezapolstat to Promote Microbiome Health

Abstract

The present invention relates to a method of using ivezapolstat to increase the health of the gut microbiome.The present invention provides a method of treating C. difficile infection while simultaneously reducing the likelihood of recurrence of C. difficile infection or preventing recurrence of C. difficile infection.The present invention also provides a method of increasing the health of the gut microbiome by increasing the number of Actinobacteria and / or Firmicutes in the gut.

Description

[Technical field]

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

[0002] The present invention relates to a method of using ivezapolstat to increase gut microbiome health. The present invention provides a method of treating Clostridioides difficile (C. difficile; formerly known as Clostridium difficile) infection (CDI) while simultaneously reducing the likelihood of recurrent C. difficile infection or preventing recurrent C. difficile infection. The present invention also provides a method of increasing gut microbiome health by increasing the number of Actinobacteria and / or Firmicutes in the gut. [Background technology]

[0003] Mucosal surfaces of the body contain complex and specialized populations of microorganisms often referred to as the microbiome or microbiota. (Mullish BH,et al.Frontline Gastroenterology 2021;12:118-127). The human gastrointestinal microbiota or microbiome is estimated to consist of up to 100 trillion microorganisms, most of which are found in the large intestine. (Kachrimanidou,Microorganisms 2020,8,200). The gastrointestinal microbiome is diverse, but in healthy adults it is primarily composed of bacteria from two major phyla: Firmicutes (gram-positive spore-forming organisms) and Bacteroidetes (gram-negative non-spore-forming organisms). These two phyla typically comprise approximately 90% of the microbiome. (Mullish BH,et al.Frontline Gastroenterology 2021;12:118-127).

[0004] In addition to Firmicutes and Bacteroidetes, the gut microbiome is also composed of Actinobacteria, Fusobacteria, Verrucomicrobia and Proteobacteria (Mullish). The Proteobacteria phylum is composed of gram-negative facultative anaerobes, and although some members of the Proteobacteria phylum are part of the healthy gut, this phylum also contains common gram-negative pathogenic commensals such as Salmonella, Shigella and Escherichia coli (Mullish). Furthermore, an increasing amount of data points to Proteobacteria as a possible microbial signature for disease. (Rizzatti, LR et al., “Proteobacteria: A Common Factor in Human Diseases”, BioMed Research International, vol. 2017, Article ID 9351507, 7 pages, 2017. https: / / doi.org / 10.1155 / 2017 / 9351507). Actinobacteria are present in large proportions in children and generally decline in overall proportion with age (replaced by Firmicutes and Actinobacteria). At birth, facultative anaerobic species such as E. coli, Staphylococcus and Streptococcus colonize the infant's gut, resulting in an anaerobic environment within the first few days of life, allowing strict anaerobes such as Bacteroides (phylum Bacteroidetes) and Bifidobacterium (phylum Actinobacteria) to proliferate.(Mueller, et al., Trends in Molecular Medicine, February 2015, Vol. 21, No. 2). Through exposure of infants to the environment and breast milk or formula over the first year of life, the gut microbiome evolves into a mature biome that resembles the adult gut microbiome. (Jangi and Lamont, JPGN, Vol. 51, No. 1, July 2010).

[0005] The gut microbiome is complex and has a mutually beneficial relationship with the host. Through this relationship, the microbiome provides many benefits to the host, including shaping the intestinal and systemic immune system, maintaining a healthy intestinal epithelium, harvesting energy from food, and protection from pathogens. (Mullish). When the composition of the microbiome is altered from its normal diversity, these beneficial physiological functions are disrupted. This is called dysbiosis. (Mullish). When the gut microbiome is in a state of dysbiosis, the microbiome contains fewer beneficial microorganisms (commensals) and more potentially harmful microorganisms (pathogenic commensals). (Mullish).

[0006] In addition to the advantages described in the previous paragraph, gut bacteria metabolize conjugated bile acids through the activity of two sets of enzymes.

[0007] The first set, bile salt hydrolase (BSH), removes conjugated taurine or conjugated glycine to generate unconjugated bile acids. After unconjugation, primary bile acids can be further metabolized by the 7α-dehydroxylation pathway to generate secondary bile acids. It has been reported that conjugated and unconjugated primary bile acids taurocholate (TCA) and cholate (CA), respectively, promote the germination of C. difficile spores, while secondary bile acids, such as lithocholate (LCA) and deoxycholate (DCA), generally inhibit the vegetative growth of C. difficile. (Qian et al., Am J Physiol Gastrointest Liver Physiol319:G227-G237,2020).

[0008] Clostridioides difficile infection (CDI) is the most common cause of healthcare-associated infections in the United States. (Magill SS,et al.Changes in prevalence of health care-associated infections in UShospitals.N Engl J Med 2018;379:1732-44). C. difficile is a normal component of the healthy gut microbiome at times, but when the microbiome becomes unbalanced, C. difficile can proliferate and cause disease (known as CDI). After C. difficile colonization, the organism produces and releases two key virulence factors, clostridial toxins A (TcdA) and B (TcdB). (Kachrimanidou,Microorganisms 2020,8,200;doi:10.3390 / microorganisms8020200). TcdA and TcdB are exotoxins that bind to human intestinal epithelial cells and cause inflammation, fluid and mucosal secretions, and damage to the intestinal mucosa. C. difficile causes a wide range of clinical symptoms, ranging from mild diarrhea to severe, life-threatening colonic perforation and toxic megacolon. In CDI, the host microbiome is prone to disruption, which is usually caused by the previous use of high-risk antibiotics. (Davis ML, et al. Multicentre derivation and validation of a simple predictive index for healthcare-associated C.difficile infection. (Clin Microbiol Infect 2018;24:1190-4). For example, treatment with broad-spectrum antibiotics can result in a near-complete loss of Bacteroidetes, a reduction in Firmicutes, and an overgrowth of Proteobacteria.These changes allow C.difficile spores to germinate, colonize, and potentially become pathogenic (Mullish). When C.difficile is activated, it produces two disease-causing toxins in the colon (Britton RA,et al.Role of the intestinal microbiota in resistance to colonization by C.difficile.Gastroenterology 2014;146:1547-53).

[0009] C. difficile colonizes approximately 60%-70% of healthy newborns and infants (Jangi 2010). However, for reasons that are not yet fully understood, these colonized infants do not show any adverse effects derived from the potent exotoxins released by this anaerobic bacterium, in contrast to older infants and adults who are prone to severe diarrhea and colitis. The organism is acquired during infancy, as in adults, from environmental contamination in the nursery or home environment. Toxigenic strains of C. difficile frequently colonize the infant intestine, with colony counts as high as those found in adults with pseudomembranous colitis, a severe manifestation of CDI (Jangi 2010). One possible reason for asymptomatic colonization in infants could be the presence and competition of other commensal flora such as Bifidobacterium (phylum Actinobacteria) and Lactobacillus (phylum Firmicutes) (Jangi 2010). Indeed, as reported by Jangi, both Bifidobacterium and Lactobacillus were able to inhibit the growth of certain C. difficile strains.

[0010] Antimicrobial therapy is a hallmark of CDI treatment, but treatment options are limited. Traditionally, metronidazole has been widely used to treat CDI, but is no longer recommended due to unacceptably high failure rates, high mortality, and cumulative toxicity compared to vancomycin. (McDonald LC,et al.Clinical practice guidelines for C.difficile infection in adults and children:2017 update by the Infectious Diseases Society of America(IDSA)and Society for Healthcare Epidemiology of America(SHEA).Clin Infect Dis 2018;66:987-94; and Stevens VW,et al.Comparative effectiveness of vancomycin and metronidazole for the prevention of recurrence and death in patients with C.difficile infection.JAMA Intern Med 2017;177:546-53).

[0011] Currently, either vancomycin or fidaxomicin are the antibiotics recommended for CDI due to their ability to kill C. difficile and to resolve clinical symptoms. (Gonzales-Luna AJ, et al., Systems biology evaluation of refractory Clostridioides difficile infection including multiple failures of fecal microbiota transplantation, Anaerobe, https: / / doi.org / 10.1016 / j.anaerobe.2021.102387). Vancomycin is recommended by the IDSA treatment guidelines, but is associated with high CDI recurrence rates and has recently been shown to increase resistance due to severe 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).Treatment with vancomycin reduces microbiome diversity of Firmicutes, Actinobacteria, and Bacteroidetes, along with characteristic Proteobacteria overgrowth. (Garey 2020). Proteobacteria overgrowth is associated with a significantly increased risk of systemic infection with multidrug-resistant (MDR) Gram-negative organisms. Vancomycin is associated with a high rate of CDI recurrence, with approximately 20–25% of patients experiencing recurrent infections after treatment cessation.(Gonzales-Luna).

[0012] Fidaxomicin has a narrower spectrum of activity than vancomycin and causes less dysbiosis over the course of treatment. Unlike vancomycin, fidaxomicin binds to C. difficile spores and prevents the growth of vegetative cells, which results in a recurrence rate that is approximately 50% lower than other treatments (Gonzales-Luna). Although recurrence rates are low with fidaxomicin, there are reports of resistance emerging via mutations in the rpoB gene (Garey 2020). Regardless of the treatment chosen, CDI recurrence rates increase with each subsequent episode of CDI, and long-term antibiotics are often required to control the disease.

[0013] Moreover, broad-spectrum antibiotic treatment has been reported to result in the loss of secondary bile acids (Qian). In addition, it has been reported that primary bile acids are increased in patients with recurrent C. difficile infection, whereas secondary bile acids are increased in healthy subjects (Qian). These results suggest that treatments that increase secondary bile acids may be effective in preventing recurrent CDI.

[0014] Thus, new therapies with different mechanisms of action against C. difficile are urgently needed. In fact, the CDC has listed C. difficile in the "urgent" category of priority pathogens for which new classes of antibiotics are needed. In particular, CDI treatments are needed that also provide an environment for the microbiome to resist relapse without the need for further treatment. CDI treatments that promote microbiome health are also needed. Summary of the Invention

[0015] The present invention provides a method for treating a C. difficile infection and simultaneously reducing the likelihood of recurrence of C. difficile infection or preventing recurrence of C. difficile infection in a subject, comprising administering an effective amount of ibezapolstat to a subject suffering from a C. difficile infection, wherein administration of the effective amount of ibezapolstat treats the C. difficile infection and simultaneously reduces the likelihood of C. difficile infection within 90 days or prevents recurrence of C. difficile infection within 90 days. Administration of ibezapolstat can be continued until clinical cure of C. difficile infection is achieved, or can be terminated when clinical cure of C. difficile infection is achieved. Administration of an effective amount of ivezapolstat may reduce the likelihood of C. difficile infection within 30 days or prevent the recurrence of C. difficile infection within 30 days.

[0016] The present invention also provides a method of promoting the growth of Actinobacteria in a subject suffering from a C. difficile infection, comprising administering an effective amount of ibezzapolstat to treat or prevent said C. difficile infection, whereby the amount of Actinobacteria in the gut microbiome of the subject is increased or the ratio of Actinobacteria to Proteobacteria is increased. The administration of ibezzapolstat may be continued until a clinical cure of the C. difficile infection is achieved, or may be terminated when a clinical cure of the C. difficile infection is achieved.

[0017] The present invention further provides a method for improving gut microbiome health, comprising administering an effective amount of ibezapolstat to a subject suffering from a C. difficile infection, wherein the proportion of bacterial phyla in the subject's gut microbiome is adjusted to a healthier balance than the subject's gut microbiome prior to administration of ibezapolstat.

[0018] The present invention further provides a method for increasing the amount of Actinobacteria in a gut microbiome, comprising administering an effective amount of Ibezapolstat to a subject in need thereof, wherein the amount of Actinobacteria is greater in the gut microbiome than the amount of Actinobacteria in the gut microbiome prior to administration of Ibezapolstat.

[0019] The present invention further provides a method for improving gut microbiome health, comprising administering an effective amount of ibezapolstat to a subject in need thereof, wherein the ratio of bacterial phyla in the subject's gut microbiome is adjusted to a healthier balance than the subject's gut microbiome prior to administration of ibezapolstat. [Brief description of the drawings]

[0020] [Figure 1] Figure 1 shows that the alpha diversity of species within the microbiome, as determined using Shannon's index, was not significantly reduced upon administration of ivezapolstat (ACX362E). In contrast, bacterial species diversity was reduced upon administration of 125 mg vancomycin. Subjects labeled placebo are subjects with healthy guts who did not receive ivezapolstat or vancomycin. Each box represents one patient who received the study drug over the 10-day period, or placebo over the 13-day study period. [Diagram 2]Figure 1 shows the effect on microbiome profile resulting from administration of 300 mg and 450 mg of ivezapolstat and 125 mg of vancomycin. Data is presented via PCoA Bray Curtis Plot. As can be seen, a different ivezapolstat microbiome profile was identified after 10 days of administration compared to vancomycin. [Diagram 3] The composition of the microbiome by bacterial phylum upon administration of 125 mg vancomycin, 300 mg ivezapolstat, and 450 mg ivezapolstat. Subjects labeled placebo are subjects with healthy gut who did not receive ivezapolstat or vancomycin. Each box represents one patient who received study drug over a 10-day period or placebo over the 13-day study period. [Figure 4A] FIG. 1 shows a Linear Effect Size Algorithm (LEfSe) summary of microbiome change from day 0 versus day 10 in subjects receiving ivezapolstat and vancomycin. Shading represents an increase or decrease in bacterial abundance at day 10 compared to baseline. [Figure 4B] FIG. 1 shows a Linear Effect Size Algorithm (LEfSe) summary of microbiome change from day 0 versus day 10 in subjects receiving ivezapolstat and vancomycin. Shading represents an increase or decrease in bacterial abundance at day 10 compared to baseline. [Figure 5A] FIG. 5B shows the changes in primary and secondary bile acids associated with administration of ibezapolstat and vancomycin, and FIG. 5B shows data in the form of the ratio of primary to secondary bile acids. [Figure 5B] FIG. 5B shows the changes in primary and secondary bile acids associated with administration of ibezapolstat and vancomycin, and FIG. 5B shows data in the form of the ratio of primary to secondary bile acids. [Figure 6]Summary estimates of alpha diversity over time by treatment group, measured by Shannon's Entropy (6A) or Simpson's Index (6B), and beta diversity measured at baseline (6C) or after at least 5 days of treatment (6D) are shown. [Figure 7] Summary changes over time in primary (7A) and secondary (7B) bile acids and the ratio of primary:secondary (7C) bile acids are shown. Values ​​represent the mean ± standard error. [Figure 8] Bile acid concentrations obtained from IBZ- and VAN-treated subjects at baseline, midpoint, and end of treatment are shown. [Figure 9] 1 shows ibezapolstat study results showing clinical cure and sustained clinical cure following administration of ibezapolstat. [Figure 10] 1 shows the PK profile of ivezapolstat against GI infections. As shown in the figure, ivezapolstat has ideal PK profile against GI infections. [Figure 11] Shown is a sample treated with Ibezapolstat that underwent a 48 hour enrichment step with taurocholate before plating on CCFA plates. [Figure 12A]

[0023] Figure 1 shows a summary estimate of alpha diversity over time following administration of ivezapolstat as measured by the Shannon Diversity Index. As shown, ivezapolstat improved alpha diversity. [Figure 12B]

[0036] Figure 1 shows a summary estimate of alpha diversity over time following administration of ivezapolstat, as measured by the Inverse Simpson Index. As shown, ivezapolstat improved alpha diversity. [Figure 13A] A chart comparing the mean proportional phylum abundance versus days of antibiotic treatment is shown. As shown in the figure, an increase in the proportion of Firmicutes was observed with Ibezapolstat treatment, with the most common increased taxon of Firmicutes being Clostridiales. [Figure 13B] A chart comparing the mean proportional class abundance versus days of antibiotic treatment is shown. As shown in the figure, an increase in the proportion of Firmicutes was observed with Ibezapolstat treatment, with the most common increased taxon of Firmicutes being Clostridiales. [Figure 13C] A chart comparing the mean proportional order abundance versus days of antibiotic treatment is shown. As shown in the figure, an increase in the proportion of Firmicutes was observed with Ibezapolstat treatment, with the most common increased taxon of Firmicutes being Clostridiales. [Figure 13D] A chart comparing the mean proportional family abundance versus days of antibiotic treatment is shown. As shown in the figure, an increase in the proportion of Firmicutes was observed with Ibezapolstat treatment, with the most common increased taxon of Firmicutes being Clostridiales. [Figure 14A] 1 shows a summary of the effect of ibezapolstat on primary bile acids over time. [Figure 14B] 1 shows a summary of the effect of ibezapolstat on secondary bile acids over time. [Figure 14C] Secondary to primary bile acid ratios are shown. Values ​​represent the mean ± SEM. [Figure 15A] 1 is a chart showing subject-specific changes in relative abundance of taxa per phylum. The chart represents one patient who received ibezapolstat for 10 days, with follow-up up to day 40. [Figure 15B] 1 is a chart showing subject-specific changes in relative abundance of taxa by class. The chart represents one patient who received ibezapolstat for 10 days, with follow-up up to day 40. [Figure 15C]1 is a chart showing subject-specific changes in relative abundance of taxa per order. The chart represents one patient who received ibezapolstat for 10 days, with follow-up up to day 40. [Figure 15D] 1 is a chart showing subject-specific changes in relative abundance of taxa per family. The chart represents one patient who received ibezapolstat for 10 days, with follow-up up to day 40. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Methods of using ivezapolstat to increase gut microbiome health are described herein. The present invention provides a method of treating C. difficile infection while simultaneously reducing the likelihood of recurrence of C. difficile infection or preventing recurrence of C. difficile infection. The present invention also provides a method of increasing gut microbiome health by increasing the number of Actinobacteria in the gut and / or increasing the proportion of Actinobacteria compared to other phyla of bacteria in the microbiome.

[0022] "C. difficile infection" or "CDI" refers to the invasion of a host animal, e.g., a mammal, by C. difficile. For example, infection can include the overgrowth of C. difficile that is normally present in or on a mammal, or the overgrowth of C. difficile that is not normally present in or on a mammal. More generally, C. difficile infection can be any situation in which the presence of C. difficile, or toxins released by C. difficile, is damaging to a host animal. An animal is "suffering" from C. difficile infection when an excess of C. difficile is present in or on the animal, or when the presence of C. difficile toxins is damaging to the animal's intestinal cells or other tissues. In one embodiment, the number of specific genera or species of C. difficile is at least 2, 4, 6 or 8 logs higher than the number found in a healthy microbiome. Alternatively, the number of C. difficile can be the same as in a healthy microbiome, but producing toxins. The presence of C. difficile infection can be characterized by the presence of toxins in stool, usually by testing the gene that produces toxin B, using PCR methods or using ELISA assays that can detect toxin proteins.

[0023] By "effective amount" is meant an amount sufficient to produce a beneficial or desired clinical or biochemical result. An effective amount can be administered one or more times. For purposes of the present invention, an effective amount is an amount of ibezapolstat that, when administered to a site of infection or potential infection, treats or prevents C. difficile infection while simultaneously increasing the amount and / or proportion of Actinobacteria and / or Firmicutes in the microbiome.

[0024] "Administration" or "administering" refers to a method of giving one or more unit doses of ibezazolstat to an animal, e.g., a mammal, such as topically, orally, intravenously, intraperitoneally, or intramuscularly. The method of administration can vary depending on 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.

[0025] By "inhibit" is meant reducing the rate of cell growth of C. difficile bacteria by at least 80%. In certain embodiments, growth may be inhibited by 90%, 95% or even 99% or more. The degree of inhibition may be ascertained, for example, by in vitro growth assays, for example, by standard liquid culture techniques. Inhibition of colony formation at a suitable MIC (minimum inhibitory concentration), for example, <100 μg / ml, more preferably <10 μg / ml, is preferred.

[0026] "Treatment" refers to an approach for obtaining beneficial or desired clinical results. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of the disease, stabilization of the disease state (i.e., not worsening), delay in progression of the disease, improvement or alleviation of the disease state, and remission (whether partial or total). "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder, as well as those in whom the disorder is to be prevented and / or recurrence is to be prevented. "Alleviating" a disease means that the extent of the disease state and / or undesirable clinical symptoms are reduced, and / or the time course of progression is slowed or prolonged compared to the situation without treatment.

[0027] "Microbiome" refers to the microorganisms within a particular environment (including the body, or a portion of the body). Preferably, the microbiome is located in the gut.

[0028] A "healthy microbiome" may be described in terms of ecological stability (i.e., ability to resist changes in community structure under stress or to rapidly return to baseline after stress-related changes) by an idealized (presumably health-related) composition or by a desirable functional profile (including metabolic and nutrient supply to the host). A healthy adult microbiome may also be characterized by a majority of bacterial species in the Firmicutes or Bacteroidetes phyla, and a minority in the Actinobacteria and Proteobacteria phyla. A healthy newborn microbiome may be characterized by a majority of bacterial species in the Bacteroidetes and Actinobacteria phyla.

[0029] "Improving gut microbiome health" means that the composition of the microbiome is dominated by bacterial species from the phyla Actinobacteria, Firmicutes, or Bacteroidetes, with a minority of Proteobacteria. Alternatively, improving gut microbiome health can mean increasing the proportion of bacterial species from the phylum Actinobacteria, as present in the gut microbiome of a healthy newborn. The subject may or may not be suffering from a C. difficile infection.

[0030] "Reducing the likelihood of C. difficile infection" means a prophylactic or treatment in which administration of ibezazolstat reduces the likelihood or rate (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90% or 95%) of a subject or patient population developing a C. difficile infection as compared to a subject or patient population not receiving ibezazolstat.

[0031] By "clinical cure" is meant that the initial infection has been resolved, preferably as measured after the subject has received treatment, about 10-12 days after diagnosis.

[0032] "Sustained clinical cure" means that the subject has had a clinical cure and has not experienced a recurrence of the infection, as measured 30-90 days after diagnosis.

[0033] By "relapse" it is meant that the subject has had a clinical cure and the infection returns within 30 to 90 days.

[0034] A healthy gut microbiome is composed of major bacterial groups called phyla. Firmicutes (gram-positive spore-forming organisms) and Bacteroidetes (gram-negative non-spore-forming organisms) are the most common and typically comprise over 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 generally decline in overall proportion with age (being replaced by Firmicutes and Actinobacteria). Proteobacteria (gram-negative facultative anaerobes) typically comprise 2-5% of the healthy microbiome. When the composition of the microbiome changes from its normal diversity, normal physiological function is disrupted (called dysbiosis). Patients suffering from C. difficile infection are in a state of dysbiosis. Dysbiosis associated with subjects with C. difficile infection includes an increase in the proportion of Proteobacteria (often called "dysbiosis") and a decrease in the number of Firmicutes and Bacteroidetes.

[0035] Ibezapolstat is 2-((3,4-dichlorobenzyl)amino)-7-(2-morpholinoethyl)-1,7-dihydro-6H-purin-6-one. Procedures for the synthesis of 1,7-dihydro-6H-purin-6-one compounds and their use in inhibiting bacterial growth are disclosed in U.S. Pat. No. 6,926,763 and U.S. Pat. No. 8,796,292, which are incorporated herein by reference. Ibezapolstat is a DNA polymerase IIIC inhibitor. Ibezapolstat has an anti-gram-positive spectrum of antibacterial activity useful for treating C. difficile infections. The mechanism of action of ivezapolstat targets gram-positive bacteria with low G+C content (fewer G and C DNA bases than A and T bases), primarily Firmicutes, including C. difficile. The DNA polymerase IIIC enzyme is essential for the replication of Gram-positive bacteria with a low G+C content and is therefore selective for Firmicutes such as C. difficile, but inactive against other host microbiota such as Actinobacteria or Bacteroidetes.

[0036] Administration of ivezapolstat results in a distinctly different microbiome profile compared to administration of vancomycin, the currently recommended treatment for C. difficile infection. For example, an increased proportion of desirable Actinobacteria and Firmicute phyla was observed in ivezapolstat-treated subjects compared to an increased number and proportion of undesirable Proteobacteria in vancomycin-treated subjects. Proteobacteria overgrowth is associated with a marked increase in the risk of systemic infection with MDR Gram-negative organisms. Thus, not only does ivezapolstat not harm the population of desirable Actinobacteria and Firmicutes in contrast to vancomycin, ivezapolstat unexpectedly increases the number and / or proportion of Actinobacteria and Firmicutes present in the microbiome. See Figures 3 and 4. By promoting the growth of healthy bacteria such as Actinobacteria and Firmicutes and not causing an increase in Proteobacteria, Ibezapolstat also provides a microbiome that prevents or reduces the likelihood of recurrent C. difficile infection.

[0037] Ibezapolstat can be formulated into pharmaceutical compositions for administration to human or animal subjects in a biologically compatible form suitable for in vivo or in vitro administration. Thus, the present invention provides pharmaceutical compositions comprising a compound of the present invention in admixture with an excipient.

[0038] The present invention provides a method for treating a C. difficile infection and simultaneously reducing the likelihood of recurrence of C. difficile infection in the subject or preventing recurrence of C. difficile infection in the subject by administering an effective amount of ibezapolstat to a subject suffering from a C. difficile infection. The administration of an effective amount of ibezapolstat treats a C. difficile infection and simultaneously reduces the likelihood of C. difficile infection within 30 to 90 days or prevents recurrence of C. difficile infection within 30 to 90 days. Preferably, administration of ibezapolstat may be continued until the C. difficile infection is clinically cured. Preferably, administration of ibezapolstat may be terminated once clinical cure is achieved.

[0039] The present invention further provides a method for promoting the growth of Actinobacteria in a subject suffering from C. difficile infection, comprising administering an effective amount of ibezapolstat to treat or prevent C. difficile infection. The amount of Actinobacteria in the gut microbiome of the subject is increased or the proportion of Actinobacteria is increased. The administration of ibezapolstat is continued until a clinical cure of C. difficile infection is achieved. Preferably, the administration of ibezapolstat is terminated when a clinical cure of C. difficile is achieved. The percentage of Actinobacteria following administration of ivezapolstat may be increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

[0040] The present invention further provides a method for improving gut microbiome health, comprising administering an effective amount of ibezzapolstat to a subject suffering from C. difficile infection. The proportion of bacterial phyla in the subject's gut microbiome is adjusted to a healthier balance than the subject's gut microbiome before administration of ibezzapolstat. For example, the proportion of Actinobacteria can be increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

[0041] The present invention also provides a method of increasing the amount of Actinobacteria in the gut microbiome, comprising administering an effective amount of ibezapolstat to a subject in need thereof, wherein the amount of Actinobacteria is greater in the gut microbiome than the amount of Actinobacteria in the gut microbiome prior to administration of ibezapolstat. The subject need not be suffering from a C. difficile infection. The present invention also provides a method of improving gut microbiome health, comprising administering an effective amount of ibezapolstat to a subject in need thereof, wherein the proportion of bacterial phyla in the gut microbiome of the subject is adjusted to a healthier balance than the gut microbiome of the person prior to administration of ibezapolstat. The subject need not be suffering from a C. difficile infection.

[0042] As shown in Figure 1, the alpha diversity of bacterial species in the microbiome was not significantly reduced upon administration of ivezapolstat. In contrast, the diversity of bacterial species was reduced upon administration of vancomycin. Subjects labeled placebo are subjects with healthy guts who were not administered ivezapolstat or vancomycin. Administration of ivezapolstat did not significantly reduce the diversity of the microbiome when compared to placebo. A reduction in bacterial diversity is undesirable as it predicts recurrence of C. difficile infection over time. As shown in Figure 2, the microbiome profiles resulting from administration of 300 mg and 450 mg of ivezapolstat differed from the microbiome profiles resulting from 125 mg of vancomycin over a 10-day period.

[0043] As shown in Figure 3, vancomycin administration increased Proteobacteria, which reduces the health and balance of the microbiome and increases the likelihood of recurrent C. difficile infection. In contrast, 300 mg of ivezapolstat increased Actinobacteria in the microbiome compared to other bacterial phyla. 450 mg of ivezapolstat also increased Actinobacteria compared to other bacterial phyla. Administration of ivezapolstat at either dose did not result in the increase in Proteobacteria seen with vancomycin administration.

[0044] Children begin to develop their gut microbiome soon after birth through exposure to a variety of bacterial species. During early infancy, facultative anaerobic species such as E. coli, Staphylococcus, and Streptococcus colonize the infant's gut, resulting in an anaerobic environment within the first few days of life, allowing strict anaerobes such as Bacteroides (phylum Bacteroidetes) and Bifidobacterium (phylum Actinobacteria) to proliferate. Also, approximately 60%-70% of healthy newborns and infants are colonized with C. difficile, often with colonization numbers as high as those of symptomatic adults with CDI. These infants do not typically experience any symptoms from this colonization. The method of the present invention results in a gut microbiome that closely resembles a healthy early infant gut microbiome, including high amounts of Actinobacteria. Bifidobacterium, a group of bacteria in the Actinobacteria phylum, inhibits the growth of C. difficile strains. Thus, the method of the present invention results in a gut microbiome that prevents or reduces the likelihood of recurrent CDI.

[0045] Ibezapolstat meets three important criteria for an ideal anti-C. difficile antibiotic: Ibezapolstat achieves high colonic concentrations with minimal systemic absorption; Ibezapolstat has potent activity against C. difficile, yet, in contrast to oral vancomycin, minimal disruption of the gut microbiome; Ibezapolstat shows potentially beneficial effects on gut bile acid metabolism.

[0046] According to the method of the present invention, ibezaporstat can be administered to a subject or patient in various forms depending on the selected administration route, as understood by those skilled in the art.For human or animal use, ibezaporstat is administered by oral, buccal, rectal and vaginal routes, or by topical administration, and is a pharmaceutical composition formulated accordingly.Preferably, ibezaporstat is administered in oral dosage form.For oral administration, the composition can be, for example, in the form of tablets, capsules, granules, liquid solutions and suspensions, but is not limited thereto.The composition can also be administered by suppository or enema.

[0047] Ibezapolstat may be administered to animals, preferably humans, alone or in combination with pharma- ceutically acceptable excipients, as described above, in a proportion determined by the solubility and chemical properties of the compound, the route of administration selected, and standard pharmaceutical practice. Ibezapolstat may be administered to adults or children. The dosage of the compounds of the present invention and / or compositions containing the compounds of the present invention may vary depending on many factors, such as the mode of administration; the age, health, and weight of the recipient; the nature and extent of symptoms; the frequency of treatment, and the type of concomitant treatment, if any; and the clearance rate of the compound in the treated animal. Those skilled in the art will be able to determine the appropriate dosage based on the above factors. The compounds of the present invention may be initially administered at a suitable dosage, which may be adjusted as necessary depending on the clinical response. In general, the compounds of the present invention may be provided in a physiologically buffered aqueous solution containing about 0.1-10% w / v of the compound, or in solid dosage form, such as tablets or capsules. A typical dosage range is about 0.01 mg / kg body weight to about 1 g / kg body weight per day. Oral dosage amounts of ibezapolstat may include amounts of about 10 mg to 1000 mg per day, preferably 100 mg to 900 mg per day, and more preferably about 150, 300, 600, or 900 mg per day.

[0048] example The foregoing description and examples are merely set forth to illustrate the present invention and are not intended to be limiting. Since modifications of the described embodiments incorporating the spirit and substance of the present invention may occur to those skilled in the art, the present invention should be broadly construed to include all modifications within the scope of the claims and their equivalents.

[0049] Example 1: Microbiome testing for Phase I healthy volunteer studies: Context: A healthy gut microbiome is composed of two major bacterial groups, called phyla. Firmicutes (gram-positive spore-forming organisms) and Bacteroidetes (gram-negative non-spore-forming organisms) are the most common. A third phylum, Proteobacteria (gram-negative facultative anaerobes), is present in low abundance but generally constitutes 2-5% of the healthy microbiome. A fourth phylum, Actinobacteria, is present in large proportions in children and generally declines in overall proportion with age (being replaced by Firmicutes and Actinobacteria). Patients suffering from C. difficile infection are in a state of dysbiosis, and such patients often have an increased proportion of Proteobacteria, e.g., an excess of Proteobacteria or "Proteobacteria dysbacteria", and reduced numbers of Firmicutes and Bacteroidetes.

[0050] Ibezapolstat Study: Using stool samples from a Phase I healthy volunteer study and shotgun metagenomic sequencing, it was demonstrated that treatment with ivezapolstat for 10 days resulted in a significantly different microbiome profile in subjects compared to subjects receiving vancomycin. The difference was that ivezapolstat-treated subjects had an increased proportion of Actinobacteria and Firmicutes phyla compared to an increased proportion of Proteobacteria in vancomycin-treated treatments.

[0051] Methods and Materials Materials: Standards of primary bile acids cholate (CA) and chenodeoxycholate (CDCA), conjugated primary bile acids glycocholate (GCA), taurocholate (TCA), glycochenodeoxycholate (GCDCA) and taurochenodeoxycholate (TCDCA), secondary bile acids lithocholate (LCA), deoxycholate (DCA), ursodeoxycholate (UDCA) and hyodeoxycholate (HDCA), and conjugated secondary bile acids glycolitocholate (GLCA), taurolithocholate (TLCA), glycodeoxycholate (GDCA) and taurodeoxycholate (TDCA) were purchased from Sigma.

[0052] Clinical trial description: 22 subjects (33% female) aged 30±8 years were enrolled. Six patients each received either vancomycin, ibezapolstat 300 mg, or ibezapolstat 450 mg, and four received placebo. As described, stool samples were collected daily as part of a recent Phase I healthy volunteer study of ascending doses of ibezapolstat (300 or 450 mg twice daily) with a vancomycin comparator arm of 125 mg four times daily and placebo. (Garey KW, et al. A randomized, double-blind, placebo-controlled, single and multiple ascending dose Phase 1 study to determine the safety, pharmacokinetics and food and faecal microbiome effects of ibezapolstat administered orally to healthy subjects. J Antimicrob Chemother 2020;75(12):3635-3643. DOI:10.1093 / jac / dkaa364.) Institutional Review Board approval was obtained (Midlands Institutional Review Board IRB #222220170383) and all volunteers signed informed consent forms prior to any study procedures. In this analysis, subjects receiving ibezazolstat 300 or 450 mg twice daily, vancomycin 125 mg four times daily, or placebo for 10 days had daily stool samples collected, when available, for follow-up periods from 0 (baseline) through days 13 and 30. Stool samples were immediately frozen at -80°C and then shipped on dry ice to the University of Houston for analysis.

[0053] Stool DNA extraction and shotgun metagenomic sequencing: Stool DNA was extracted using the DNAeasy Power Soil Pro kit (Qiagen, Cat. No. 1288-100) on a QiaCube automated DNA extraction system as previously described. (Garey KW, et al., J Antimicrob Chemother 2020;75(12):3635-3643. DOI:10.1093 / jac / dkaa364.) Shotgun metagenomic sequencing was performed at the University of Houston Sequencing and Gene Editing Core (Houston, TX USA) using the Nextera DNA Flex Library Prep Kit for DNA library preparation and the Illumina NextSeq 500 platform for sequencing. CLC Genomic Workbench version 12 (Qiagen) was used for metagenomic assembly and for generating abundance tables.

[0054] Extraction of bile acids from stool samples: Stool samples were aliquoted and weighed (ranging from about 10 to about 150 mg), and each aliquot was thoroughly mixed with 1 ml of 100% methanol containing internal standards (LCA-d5 and CA-d5, 200 μg / L) by vortexing and sonication. The mixture was left at 4 °C overnight, centrifuged at 10,000 g for 3 min, and the supernatant was transferred to a new tube and diluted 10-fold with pure water. The diluted supernatant was then applied to a preconditioned Sep-Pak C18 Classic Cartridge or Waters Corp Oasis HLB 96-well Plate (Waters, USA). After washing with 5% methanol, the bile acid fraction was eluted with 100% methanol. The eluate was dried under nitrogen, resuspended in 2 ml of methanol / water (1:1, vol / vol), and stored at -20 °C until further analysis.

[0055] Bile acids analysis: Bile acids were quantified using targeted liquid chromatography-mass spectrometry (LC-MS) performed with a QTRAP 5500 mass spectrometer (Sciex, Framingham, MA, USA) adapted from a previously described method. (Scherer M, et al. Rapid quantification of bile acids and their conjugates in serum by liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2009;877(30):3920-5. DOI:10.1016 / j.jchromb.2009.09.038.) In summary, a chromatographic separation between bile acids of similar mass and chemical structure was achieved using a gradient method with two mobile phases (solvent A: methanol-water (1:1, vol / vol) containing 10 mM ammonium acetate and 0.1% (wt / vol) ammonium hydroxide (pH 9)); solvent B: methanol containing 10 mM ammonium acetate and 0.1% (wt / vol) ammonium hydroxide (pH 9)) on a C18 column (Phenomenex, Torrance, CA, USA). Quantitation of each type of bile acid was calculated from standard curves constructed using unlabeled and stable isotope-labeled standards of bile acids. Bile acid concentrations were normalized by the corresponding sample weights.

[0056] statistical analysis R software was used to generate subject-specific and summary changes in bacterial taxa and alpha diversity. Linear regression models were constructed to evaluate proportional taxon differences at the phylum, class, order, and family levels over time for subjects receiving vancomycin or ibezzapolstat, normalizing to taxa present in at least 5 percent of all samples. Linear regression models were also constructed to evaluate daily changes in alpha diversity measures (Shannon, Simpson, and Pielous) over time for subjects receiving vancomycin or ibezzapolstat. A linear effect size (LEfSe) algorithm was used to visualize and identify significant differences in microbiota composition between baseline and day 10 samples. (Segata N, et al. Metagenomic biomarker discovery and explanation. Genome Biol 2011;12(6):R60. DOI:10.1186 / gb-2011-12-6-r60.) Linear regression models were constructed to evaluate the change in primary and secondary bile acids over time and the ratio of primary to secondary bile acids over time from subjects receiving vancomycin or ibezazolstat. All linear regression models used placebo results as baseline values, controlling for subject age, weight, and sex. All statistical analyses were performed using SAS Vers 9.4 (Sas Institute, Cary NC) or R. To account for multiple analyses per objective, p values ​​as low as p<0.005 were considered statistically significant unless otherwise stated to limit false discovery rates. (Korthauer K,et al.A practical guide to methods controlling false discoveries in computational biology.Genome Biol 2019;20(1):118.DOI:10.1186 / s13059-019-1716-1.)

[0057] Alpha and Beta Diversity Alpha and beta diversity: Alpha diversity tests for the diversity of bacterial species within a sample, whereas beta diversity tests for the difference in diversity between samples. Using the method described by Gonzales-Luna (Systems biology evaluation of refractory Clostridioides difficile infection including multiple failures of fecal microbiota transplantation. Anaerobe 2021:102387. DOI:10.1016 / j.anaerobe.2021.102387), alpha and beta diversity were assessed in samples from healthy subjects treated with ivezapolstat and compared to subjects treated with vancomycin.

[0058] 16S Ribosomal RNA (rRNA) Gene Sequencing: 16S rRNA sequencing was performed to characterize microbial taxonomy as described in Gonzales-Luna 2021. The V3-V4 region of the 16S rRNA gene was sequenced to assess gut microbiome community structure using an Illumina-based sequencing platform with a minimum of 15,000 reads per sample. Quality-filtered sequence reads with at least 97% similarity were clustered into Operational Taxonomic Units (OTUs) and representative sequences from each OTU were assigned taxonomic identity at the species level by searching against the NCBI 16S rRNA sequence database (published date September 1, 2018) using the NCBI BLAST+ package v2.8.1 2018. Microbial diversity indices were calculated using QIIME v1.9.0, where species richness and phylogenetic distance represent α-diversity, and Bray-Curtis and Weighted Unifrac represent β-diversity. To visualize the results, the R platform and GraphPad Prism 7.0 (San Diego, CA) were used.

[0059] Shotgun metagenomic sequencing: DNA extracted from fecal samples previously used for 16S rRNA sequencing was shotgun metagenomic sequenced using an Illumina-based platform for the analysis of microbiome functional genes. Functional gene profiling of shotgun metagenomics was performed using the HUMAnN2 v0.11.2 pipeline. 35 Preprocessing steps included quality filtering of sequencing reads, followed by screening and removal of contaminating host (human) reads. Trimmomatic v0.38 was used for filtering and trimming of raw sequence data with default cutoff settings. Reads were searched against the human genome database in paired-end mode using the bowtie2 algorithm and discarded if they were mapped to the database. To obtain gene family profiles, these quality-controlled metagenomic sequences were first searched against a nucleotide database (ChocoPhlAn) using bowtie2 and then against a protein database (UniRef90) using diamond. All identified gene families were annotated using UniRef90 and pathways were annotated using MetaCyc identifiers. Gene family profiles were used to generate alpha and beta diversity.

[0060] The results of the alpha diversity analysis are shown in Figure 1. In the figure, the top row of plots represents subjects receiving vancomycin treatment; the second row of plots represents subjects receiving 300 mg of ibezapolstat treatment; and the third row of plots represents subjects receiving 450 mg of ibezapolstat treatment. The fourth row represents subjects receiving placebo. As can be seen in this figure, treatment with ibezapolstat results in less overall change in the alpha diversity of the biome over the course of treatment compared to vancomycin.

[0061] The results of the beta diversity analysis are shown in Figure 2. In the figure, the plot on the left shows the baseline diversity in subjects before treatment. The plot on the right shows the beta diversity of subjects receiving either vancomycin or ibezzapolstat. Subjects receiving ibezzapolstat have a beta diversity that stays around the same y-axis, whereas vancomycin subjects show a much different beta diversity. The results of this study demonstrate that gut diversity is different following administration of ibezzapolstat compared to vancomycin.

[0062] Microbiome Analysis The microbiome of healthy subjects receiving either ibezapolstat or vancomycin treatment was analyzed using methods described by Gonzales-Luna (Systems biology evaluation of refractory Clostridioides difficile infection including multiple failures of fecal microbiota transplantation. Anaerobe 2021:102387. DOI:10.1016 / j.anaerobe.2021.102387) and Segata et al. (Metagenomic biomarker discovery and explanation. Genome Biol 2011;12(6):R60. DOI:10.1186 / gb-2011-12-6-r60).

[0063] Using the above sequencing, we analyzed the time course of bacterial phyla in subjects receiving either ibezzapolstat or vancomycin. The results of this analysis are shown in Figure 3. As can be seen from the results of this analysis, treatment with vancomycin results in a "dysbiosis" of Proteobacteria (shown in dark grey in the plot). Also evident from this analysis is that treatment with ibezzapolstat results in a higher proportion of Actinobacteria.

[0064] We analyzed data from healthy volunteers treated with either ibezapolstat or vancomycin using the Linear Discriminant Analysis Effect Size (LEfSe) method, which allows for high-dimensional class comparisons with a particular focus on metagenomic analysis. This method is described in Segata et al. (Metagenomic biomarker discovery and explanation. Genome Biol 2011;12(6):R60. DOI:10.1186 / gb-2011-12-6-r60). The results of this analysis are shown in Figure 4. The figure shows the changes in the microbiome on day 0 versus day 10 in subjects administered either ibezapolstat or vancomycin. Shading represents a decrease in bacterial abundance, or an increase in abundance, on day 10 compared to baseline. As evident in the LEfSe plot, ibezapolstat caused much less change to the overall microbiome on day 10 of treatment compared to vancomycin on day 10.

[0065] The baseline microbiota did not differ at baseline (day 0 samples) for any of the study arms. The daily changes over time in individual phyla and Shannon's index alpha diversity for subjects receiving ivezapolstat, vancomycin or placebo are shown in Figures 1 and 3. Differences between individual phyla were evident. However, in general, subjects receiving vancomycin had an increased proportion of Proteobacteria or Fusobacteria, whereas subjects receiving ivezapolstat had a consistent increase in the proportion of Actinobacteria. In general, treatment of individual subjects receiving either ivezapolstat or vancomycin resulted in a decrease in alpha diversity compared to placebo. A statistical analysis of the alpha diversity changes over time is shown below in Table 1. Using three separate alpha diversity indices (Shannon, Simpson, and Pielous), ivezapolstat 450 mg and vancomycin showed statistically significant changes in alpha diversity over time compared to placebo. ivezapolstat 300 mg showed no statistically significant changes compared to placebo. Summary measures of alpha diversity change (Shannon) over time by treatment group are shown in Figure 6. Changes in beta diversity confirmed that the microbiota was significantly different between study groups (Figure 6). Using principal coordinate analysis, baseline samples were similar across all study groups, but distinct ellipses representing 95% confidence for each cluster were significantly different for vancomycin-treated subjects compared to either dose of ivezapolstat samples or placebo samples. Cladograms at baseline compared to end of treatment, generated by the LEfSe algorithm, are shown in Figure 4. Vancomycin had an even broader effect on the microbiome, including significantly lower proportions of most taxa, with the exception of an increased proportion of Gammaproteobacteria.Ibezapolstat showed a decrease in the proportion of Clostridiales and an increase in the proportion of certain species of Enterobacteriaceae, and Bifidobacteriaceae. The changes in bacterial taxa at the phylum, class, order and family levels are shown in Table 2 below. [Table 1] [Table 2-1] [Table 2-2]

[0066] bile acid analysis To further analyze the changes in bile acids associated with the use of ibezzapolstat, Scherer M,et al. (Rapid quantification of bile acids and their conjugates in serum by liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2009;877(30):3920-5. DOI:10.1016 / j.jchromb.2009.09.038) and Qian X et al. (Ridinilazole, a narrow spectrum antibiotic for treatment of Clostridioides difficile infection, enhances preservation of microbiota-dependent bile acids. Am J Physiol Gastrointest Liver Physiol 2020;319(2):G227-G237.DOI:10.1152 / ajpgi.00046.2020), an LC-MS-MS method was developed for this study.

[0067] Seventeen baseline samples, five samples from 17 days, and ten samples from 14 days were available for bile acid analysis. Concentrations of bile acids for each drug and time period are shown in Figure 8. Baseline samples were similar for all study groups and contained primarily (>95%) secondary bile acids. Exposure to all study drugs increased primary bile acids and decreased secondary bile acids (Figure 7). Using linear regression analysis controlling for subject demographics, vancomycin was associated with significant increases in primary bile acids and primary:secondary bile acid ratios. Similar effects were seen with ivezapolstat 450 mg, although these results were not statistically significant (Table 1 above).

[0068] The results of this analysis are shown in Figures 5A and 5B. The plots on the left show the changes in primary bile acids over the course of treatment with either ibezapolstat or vancomycin. The plots on the right show the changes in secondary bile acids over the course of treatment with either ibezapolstat or vancomycin. As is evident from these results, treatment with vancomycin causes a large increase in the primary bile acids as well as a large decrease in the secondary bile acids. In contrast, ibezapolstat does not cause a significant change in the amount of primary bile acids and does not cause the same large decrease in secondary bile acids.

[0069] Correlation between microbiota and bile acid changes Correlations between family taxa and primary and secondary bile acid concentrations are shown in Table 3. Enterobacteriaceae was most highly correlated with primary bile acid concentrations (r: 0.63; p < 0.0001), while Ruminococcaceae was negatively correlated with primary bile acid concentrations (r: -0.37; p = 0.0025). Ruminococcaceae was positively correlated with secondary bile acid concentrations (r: 0.44; p = 0.0002). Pseudomonadaceae was also positively correlated with secondary bile acid concentrations (r: 0.38; p = 0.0017). [Table 3]

[0070] Key metagenomic findings in this study were a consistent reduction in the class Clostridia with both antibiotics, but an expansion of the class Actinobacteria in ibezapolstat-treated subjects, and an expansion of the class Gammaproteobacteria, order Enterobacterales, and family Enterobacteriaceae in vancomycin-treated subjects.Within the phylum Firmicutes, vancomycin was also associated with an increase in the proportion of Bacilli taxa.

[0071] Example 2: Microbiome data from a phase 2a clinical trial of ivezapolstat for CDI Phase 2 Clinical Trials: A phase 2 clinical trial is designed to evaluate ibezapolstat in the treatment of CDI.

[0072] Phase 2a of the study was an open-label cohort of 10 subjects from a US study center. In this cohort, 10 patients with mild / moderate C. difficile-induced diarrhea diagnosed via toxin EIA+ were treated with ibezapolstat 450 mg orally twice daily for 10 days. All patients were followed for recurrence for 28±2 days. Stool was collected during the course of treatment and at the follow-up period. Patients' stool samples were evaluated for C. difficile cultures and microbiome changes. The study demonstrated 100% clinical cure at day 12 and 100% sustained clinical cure at day 38. Favorable microbiome changes included overgrowth of species from the phyla Actinobacteria and Firmicutes during treatment. These findings demonstrate a beneficial effect on bile acid metabolism and further support that microbiome effects may portend favorable patient outcomes, including lower recurrence rates.

[0073] Infection was cleared 100%, there was no recurrence of infection (100%), and the adverse event profile was tolerable.

[0074] method: Safety assessment Safety assessments included AE assessment, physical examination, vital signs, laboratory tests (chemistry, hematology, and urinalysis), and electrocardiogram. Safety endpoints including the nature, frequency, and severity of AEs were recorded for all subjects. AEs were assessed at each visit beginning at enrollment and were classified according to the Medical Dictionary for Regulatory Activities (MedDRA version 15.0). AE severity (mild, moderate, or severe) and causality (unrelated, possibly related, or probably related to study drug) were assessed by the investigators at each site.

[0075] Penalty rating Plasma levels were measured 2 and 4 hours after the first daily ibezapolstat dose on days 1, 5, and 10. Fecal samples were collected at baseline and after daily ibezapolstat doses on days 1 through 10. Plasma and fecal concentrations were assayed by AltaSciences (Laval, Quebec, Canada), and PK analyses were performed by Learn and Confirm Inc. (Montreal, Quebec, Canada).

[0076] microbiology Stool samples were cultured for C. difficile growth on selective cycloserine-cefoxitin fructose agar (CCFA) at 37°C under anaerobic conditions for 48 h (Gonzales-Luna). Isolates were determined to be C. difficile based on growth and morphology and confirmed by PCR for C. difficile toxins and tpi genes. C. difficile was strain typed using a PCR-based ribotyping method as previously described. (Gonzales-Luna AJ, Carlson TJ, Dotson KM, et al. PCR ribotypes of Clostridioides difficile across Texas from 2011 to 2018 including emergence of ribotype 255. Emerg Microbes Infect 2020;9(1):341-7.) Minimum inhibitory concentrations (MICs) were determined for ibezapolstat 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).)

[0077] Microbiome and bile acid assessment Fecal samples for microbiome analysis were collected daily during ivezapolstat administration and on days 2, 10, 20, and 28 after EOT. Fecal DNA extraction was performed via the Qiagen DNeasy PowerSoil Pro Kit (Qiagen, Cat. No. 12888-100) on a QIAcube automated DNA extraction system (Qiagen) according to the instructions. Microbiome characterization was performed by sequencing the V1-V3 region of the 16S rRNA gene using the MiSeq system (Illumina, San Diego, CA, USA). (Fadrosh DW, Ma B, Gajer P, et al. An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform. Microbiome 2014;2(1):6; Walker JN, Hanson BM, Pinkner CL, et al. Insights into the Microbiome of Breast Implants and Periprosthetic Tissue in Breast Implant-Associated Anaplastic Large Cell Lymphoma. Sci Rep 2019;9(1):10393.) Barcoded primers were used to amplify each sample, resulting in a unique sequence identifier tagged to each individual sample library. Genomic DNA (gDNA) was normalized before PCR and PCR products were normalized before pooling. Illumina-based sequencing yielded >15,000 reads per sample. Bile acids were quantified using targeted liquid chromatography-mass spectrometry (LC-MS) performed with a QTRAP 5500 mass spectrometer (Sciex, Framingham, MA, USA) adapted from a previously described method.(Scherer M, Gnewuch C, Schmitz G, Liebisch G. Rapid quantification of bile acids and their conjugates in serum by liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2009;877(30):3920-5.) Bile acid levels were normalized by the corresponding stool sample weight.

[0078] Efficacy evaluation The primary efficacy outcome measure was clinical cure at EOT, defined as resolution of diarrhea within the 24-h period before EOT and maintained for at least 48 h after EOT. SCC was defined as clinical cure without recurrence of CDI within 28 (± 2) days after EOT.

[0079] statistical analysis An intent-to-treat analysis was performed for patients receiving at least one dose of ivezapolstat. Descriptive statistics were calculated for efficacy, safety / tolerability, and PK data generated using SAS version 9.4 software (SAS Institute, Inc Cary, NC, USA). Microbiome summary plots and data visualizations were created using R software version 4.1.1 (R Core Team 2021, Vienna, Austria). (R Core Team (2013). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria.) Alpha diversity was assessed for each sample using the VeganR package version 2.4-2 using the Shannon Diversity Index and Inverse Simpson Index. Linear regression models were used to compare differences in alpha diversity (Shannon Diversity Index and Inverse Simpson Index) and bile acids at baseline compared with during or after treatment. Proportional changes in bacterial taxa over the 10-day dosing interval were calculated using a linear regression model for taxa with a proportional change of at least 1 percent over the study period. A p-value <0.05 was considered significant.

[0080] Phase 2a Clinical Trial Results: Phase 2a data demonstrated complete eradication of colonic C. difficile by day 3 of treatment with ibezapolstat, as well as observed overgrowth of healthy gut microbiota, Actinobacteria species, and Firmicute species during and after treatment. Additionally, data demonstrated an increase in the proportion of healthy microbiota, including Clostridiales taxa, which are known to metabolize primary bile acids to secondary bile acids via the 7α-dehydroxylation pathway. (Ridlon JM, Kang DJ, Hylemon PB. Bile salt biotransformations by human intestinal bacteria. J Lipid Res 2006;47(2):241-59). These data demonstrate an increase in secondary bile acid concentrations during and after ibezapolstat therapy that correlates with colonization resistance to C. difficile. Furthermore, the reduction in primary bile acids and favorable increase in the ratio of secondary to primary bile acids indicates that ibezapolstat may reduce the likelihood of CDI recurrence when compared with vancomycin.

[0081] patient Ten patients aged 49 (± 15) years (50% female; 100% Caucasian; 80% Hispanic or Latino ethnicity) were enrolled. All 10 patients received ivezapolstat, and all 10 patients completed the study. The median number of unformed stools within 24 hours before starting treatment was 4 (range: 3-10). Two of the 10 patients had received antibiotic treatment with either metronidazole or vancomycin for less than 24 hours before starting ivezapolstat. No patients were hospitalized before or after enrollment.

[0082] safety A summary of AEs is shown in Table 4. Seven AEs were reported in 4 of the 10 patients, with 4 of these events occurring in 1 subject. None of the events were serious AEs. AE severity was mild (n=2), moderate (n=4), and severe (n=1; non-drug related migraine). The most common AEs were headache (n=2) or nausea (n=2). Both episodes of nausea were considered "probably related" to the study drug by the investigator. No treatment was required for these AEs, and no AEs necessitated changes in the study drug schedule or discontinuation. All AEs resolved by the end of the study.

[0083] PK result Ibezapolstat plasma levels 2-4 hours post-dose ranged from 233-578ng / mL, with higher concentrations observed during the 4 hour post-dose period (range: 373-578ng / mL) compared to the 2 hour post-dose period (range: 234-299ng / mL). By day 3 of treatment, ibezazolstat stool concentrations averaged 416±494μg / g stool and increased to >1,000μg / g stool by days 8-10 of treatment. Concentrations averaged 535±748μg / g stool 2 days after EOT. In three of four stool samples collected on day 38, ibezazolstat stool concentrations remained detectable (136±161μg / g stool). Total stool and plasma PK data are shown in Figure 10. Ibezapolstat achieved high stool and plasma concentrations not exceeding 1ug / mL.

[0084] Microbiological results Seven baseline stool samples were available for microbiology testing, of which six (86%) grew toxigenic C. difficile. Stool samples obtained from all other collection days (range 7–9 samples / day) did not grow C. difficile. Ribotypes identified included F078-126 (n=2), F014-020 (n=2), F106 (n=1), and FP435 (n=1). MICs for ivezapolstat were 0.25 (n=1), 0.5 (n=3), or 1.0 (n=1) ug / mL.

[0085] Microbiome and Bile Acid Results Using both the Inverse Simpson Index and the Shannon Index, a rapid increase in alpha diversity was observed from the baseline sample (Figure 12). Compared to baseline, Inverse Simpson Index diversity increased by 0.14 ± 0.056 points during ibezapolstat therapy (p = 0.017) and by 0.22 ± 0.10 points after EOT (p = 0.0033).

[0086] Similar results were observed using the Shannon Diversity Index. Diversity increased by 0.98 ± 0.48 points during ivezapolstat therapy (p = 0.049) and by 1.7 ± 0.87 points after completion of treatment (p = 0.043) compared to baseline. Changes in taxa during and after ivezapolstat therapy are shown in Figure 13. Most commonly, a proportional decrease in the phylum Bacteroidetes was observed (-10.0 ± 4.8%; p = 0.043), due to a decrease in the proportion of Bacteroidia class taxa (-10.0 ± 4.8%) and Flavobacteriaceae family taxa (-8.8 ± 4.8%). Most commonly, an increase in the proportion of the phylum Firmicutes was observed (+14.7 ± 5.4%; p = 0.009), due to an increase in the proportion of Lachnospiraceae (+12.7 ± 6.0%) and Ruminococcaceae (+2.8 ± 2.7%). Other Firmicutes showed a decrease in proportion, most notably in the orders Bacillales (-4.4 ± 2.3%) and Lactobacillales (-3.7 ± 2.2%). Abundance tables for individual patients are shown in Figure 15.

[0087] The results of the bile acid analysis are shown in Figure 14. Compared to baseline, total primary acids in the stool decreased by 40.1 ± 9.6 ng / mg stool during treatment (p = 0.0002) and by 40.5 ± 14.1 ng / mg stool after completion of treatment (p = 0.0066). Compared to baseline, total secondary bile acids increased by 65.6 ± 146.7 ng / mg stool during treatment (p = 0.66) and by 97.5 ± 215.4 ng / mg stool after completion of treatment (p = 0.65). [Table 4]

Claims

1. A pharmaceutical composition comprising ibezapolstat for treating Clostridioides difficile infection while simultaneously reducing the likelihood of recurrence of Clostridioides difficile infection in a subject or preventing recurrence of Clostridioides difficile infection in the subject, wherein said Clostridioides difficile infection is treated while simultaneously reducing the likelihood of Clostridioides difficile infection within 90 days or preventing recurrence of Clostridioides difficile infection within 90 days.

2. The pharmaceutical composition according to claim 1, wherein the administration of ibezapolstat is continued until clinical cure of the Clostridioides difficile infection is achieved or there is no recurrence for 40 to 90 days.

3. A pharmaceutical composition comprising ibezapolstat for promoting the growth of Actinobacteria in a subject suffering from Clostridioides difficile infection, increasing the relative abundance of Actinobacteria in the gut microbiota of the subject or increasing the proportion of Actinobacteria compared to Proteobacteria, and increasing the total secondary bile acids in the feces of the subject.

4. The pharmaceutical composition according to claim 3, wherein the administration of ibezapolstat is continued until clinical cure of the Clostridioides difficile infection is achieved.

5. A pharmaceutical composition comprising ibezapolstat for improving the health of the gut microbiota of a subject suffering from Clostridioides difficile infection, adjusting the proportion of bacterial phyla in the gut microbiota of the subject to a healthier balance than the gut microbiota of the subject before administration of ibezapolstat. A pharmaceutical composition for increasing the total secondary bile acids in the feces of the subject. Claim 6 A pharmaceutical composition comprising evesapostat for increasing the relative abundance of Actinobacteria in the gut microbiota, wherein the relative abundance of Actinobacteria in the gut microbiota is increased compared to the amount of Actinobacteria in the gut microbiota before administration of the evesapostat, A pharmaceutical composition for increasing the total secondary bile acids in the feces of the subject. Claim 7 A pharmaceutical composition comprising evesapostat for improving the health of the gut microbiota, wherein the relative abundance and / or ratio of the bacterial phyla in the gut microbiota of the subject is adjusted to a healthier balance compared to the gut microbiota of the subject before administration of the evesapostat, A pharmaceutical composition for increasing the total secondary bile acids in the feces of the subject. Claim 8. The pharmaceutical composition according to claim 5 or 7, wherein the relative abundance and / or ratio of the bacterial phyla is maintained for 10 to 40 days. Claim 9. The pharmaceutical composition according to claim 6, wherein the relative abundance of Actinobacteria in the gut microbiota is made similar to the relative abundance of Actinobacteria found in the gut microbiota of healthy children.

10. Making the relative abundance and / or ratio of the genera Firmicutes, Bacteroidetes, Fusobacteria, Verrucomicrobia, and / or Proteobacteria in the intestinal microbiota into a healthy balance, preferably, decreasing the relative abundance and / or ratio of Clostridium in the intestinal microbiota, increasing the relative abundance and / or ratio of at least one of Enterobacteriaceae and / or Bifidobacteria, or During and / or after administration of the pharmaceutical composition, increasing the relative abundance and / or ratio of Firmicutes from the taxon Clostridiales, preferably, the increase in the taxon Clostridiales includes an increase in the relative abundance and / or ratio of Lachnospiraceae and / or Ruminococcaceae, the pharmaceutical composition according to claim 5.

11. Increasing the relative abundance of species of the phyla Actinobacteria and Firmicutes during and / or after administration of the pharmaceutical composition, preferably, the increase in the relative abundance includes an increase in the relative abundance and / or ratio of Bifidobacterium and / or Lactobacillus, more preferably, the increase in the relative abundance and / or ratio of Bifidobacterium in the intestinal microbiota inhibits the growth of C. difficile strains and / or prevents or reduces the likelihood of recurrence of C. difficile infection, the pharmaceutical composition according to claim 5.

12. During and / or after administration of the ibezapolstat, decreasing the relative abundance and / or ratio of the order Bacillales and / or Lactobacillales, or The relative abundance and / or ratio of microorganisms belonging to the phylum Firmicutes and / or Bacteroidetes is increased compared to the relative abundance and / or ratio of microorganisms belonging to the phylum Proteobacteria, or The relative abundance and / or ratio of microorganisms belonging to the phylum Firmicutes and / or Bacteroidetes is increased compared to the relative abundance and / or ratio of Proteobacteria, or The relative abundance and / or ratio of microorganisms belonging to the phylum Firmicutes and Bacteroidetes account for up to 90% of the gut microbiota. The pharmaceutical composition according to claim 5.

13. Administration of the evesapostat increases the diversity of the gut microbiota of Firmicutes, Actinobacteria, and Bacteroidetes, and administration of the evesapostat reduces or eliminates the overgrowth of Proteobacteria, or Administration of the evesapostat increases the relative abundance and / or ratio of the phylum Actinobacteria and Firmicutes compared to the relative abundance and / or ratio of the phylum Proteobacteria, or Administration of the pharmaceutical composition reduces the relative abundance of the phylum Bacteroidetes in the gut microbiota, and preferably, the relative decrease of the Bacteroidetes is due to a decrease in the ratio of taxa of the class Bacteroidia and taxa of the family Flavobacteriaceae, or Administration of said evesapostat proportionally increases the relative abundance of the healthy microbiota, and the proportion of the healthy microbiota includes an increase in the microbiota that metabolizes primary bile acids to secondary bile acids via the 7α-dehydroxylation pathway, or The pharmaceutical composition according to claim 5, wherein administration of said evesapostat reduces the amount of primary bile acids and / or increases the ratio of secondary bile acids to primary bile acids, thereby reducing the risk of recurrence of Clostridioides difficile infection.

14. During and / or after administration of said pharmaceutical composition, Gram-negative aerobic-anaerobic bacteria account for about 2-5% of the microbiome, preferably the Gram-negative aerobic-anaerobic bacteria are Proteobacteria, or The pharmaceutical composition according to claim 5, wherein the amount of primary bile acids present in the intestine before administration of said pharmaceutical composition is similar to the amount of primary bile acids present in the intestine after administration of said pharmaceutical composition.

15. During and after administration of said pharmaceutical composition, the concentration of secondary bile acids in the intestine increases, thereby improving resistance to Clostridioides difficile, or The amount of total primary bile acids in the feces of the subject decreases during and after administration of said evesapostat, or The pharmaceutical composition according to claim 5, wherein the amount of total secondary bile acids in the feces of the subject increases during and after administration of said evesapostat.

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