Pharmaceutical composition for treating or preventing hyperammonemia

Abstract

To provide a pharmaceutical composition for treating or preventing hyperammonemia and hepatic encephalopathy.SOLUTION: A pharmaceutical composition for treating or preventing hyperammonemia contains rifaximin and conjugated bile acid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a pharmaceutical composition for treating or preventing hyperammonemia. [Background technology]

[0002] Currently, treatments for hepatic encephalopathy (hyperammonemia) associated with liver cirrhosis include the administration of branched-chain amino acids, urea cycle activators, and non-absorbable synthetic disaccharides. Furthermore, if these treatments are not effective, treatment with the poorly absorbable antibiotic rifaximin (sometimes referred to as "RFX" in this specification) is performed, as ammonia production is largely carried out by the intestinal bacterial flora (Patent Document 1).

[0003] However, there is a problem that there is no effective treatment for patients with hepatic encephalopathy (hyperammonemia) for whom rifaximin administration does not significantly improve the condition. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 57-11987 Summary of the Invention [Problem to be solved by the invention]

[0005] An objective of the present disclosure is to provide a pharmaceutical composition for treating or preventing hyperammonemia. [Means for solving the problem]

[0006] The present inventors have conducted extensive research to solve the above problems and have discovered that Streptococcus salivarius is the causative bacterium of hyperammonemia, that Ruminococcus gnavus enhances the urease activity of other ammonia-producing bacteria, and that the sensitivity of S. salivarius and R. gnavus to rifaximin is enhanced in the presence of taurodeoxycholic acid, and have made further improvements.

[0007] The present disclosure encompasses, for example, the subject matter described in the following sections: Section 1. A pharmaceutical composition for treating or preventing hyperammonemia, comprising rifaximin and conjugated bile acids. Section 2. A pharmaceutical composition for treating or preventing hepatic encephalopathy, comprising rifaximin and a conjugated bile acid. Section 3. 1. A pharmaceutical composition comprising a conjugated bile acid, A pharmaceutical composition for use in a human being who is or has been administered rifaximin. Section 4. Item 4. The pharmaceutical composition according to any one of Items 1 to 3, wherein the conjugated bile acid is taurodeoxycholic acid or glycodeoxycholic acid. Section 5. Conjugated bile acids, including An agent that enhances the therapeutic or preventive effect of rifaximin on hyperammonemia or hepatic encephalopathy. [Effects of the Invention]

[0008] Pharmaceutical compositions for the treatment or prevention of hyperammonemia are provided. [Brief explanation of the drawings]

[0009] [Figure 1] A diagram of the breakdown of clinical samples is shown. [Figure 2A] Comparison of intestinal microbiota composition (comparison between healthy individuals and patients with hepatic encephalopathy) is shown. [Figure 2B] Comparison of intestinal microbiota composition (comparison between healthy individuals and patients with hepatic encephalopathy) is shown. [Figure 2C] The results of an analysis of the bacterial genera that are common in the HE (Hepatic Encephalopathy) group are shown below. [Figure 2D] The results of an analysis of the bacterial species that are more prevalent in the HE (Hepatic Encephalopathy) group compared with healthy controls (HC) are shown below. [Figure 3A] Quantitative results of S. salivarius in human fecal DNA samples are shown. [Figure 3B] Correlation between the abundance of Streptococcus salivarius 16S rRNA gene DNA and UreC DNA is shown. [Figure 3C] 1 shows the results of a urease test using S. salivarius NCTC 7366 strain and S. salivarius ATCC 25975 strain. [Figure 4A] 1 shows the results of measuring the amount of ammonia in mouse plasma. [Figure 4B] Quantitative results of S. salivarius in mouse fecal DNA samples (left) and abundance of UreC (a subunit of the urease enzyme) are shown. [Figure 5] The amount of S. salivarius present in the culture medium at each concentration of rifaximin (RFX) is shown. [Figure 6] Changes in plasma ammonia levels (A), total fecal bacterial load (B), and relative abundance of S. salivarius (C) before and 4 weeks after rifaximin administration are shown. [Figure 7] The results of quantification of S. salivarius by UreC quantitative PCR before and 4 weeks after rifaximin administration are shown. [Figure 8A] The results of an analysis of bacterial species that are common in non-responders are shown below. [Figure 8B] The results of quantification of Ruminococcus gnavus before and 4 weeks after rifaximin administration are shown. [Figure 8C] The results of urease tests using R. gnavus (RG) or recombinant urease are shown. [Figure 9A] The results of urease tests using various strains are shown. [Figure 9B] The results of the urease test using recombinant urease and R. gnavus are shown. [Figure 10A] Heat maps of plasma bile concentrations in responder patients (R) and non-responder patients (NR) before RFX administration are shown. [Figure 10B] The figures show the amounts of CA (cholic acid), DCA (deoxycholic acid), TDCA (taurodeoxycholic acid), and GDCA (glycodeoxycholic acid) in the plasma of responder patients (R) and non-responder patients (NR) before RFX administration. [Figure 11] The abundance of R. gnavus (left) and S. salivarius (right) in the presence of RFX is shown with and without the addition of TDCA (R. gnavus: 5 mM, S. salivarius: 1 mM). [Figure 12] The figures show the abundance of R. gnavus and S. salivarius bacteria in the culture medium at various DCA concentrations when DCA was added. [Figure 13] The figures show the abundance of R. gnavus and S. salivarius bacteria in the culture medium at various RFX concentrations when DCA and RFX were added. DETAILED DESCRIPTION OF THE INVENTION

[0010] Each embodiment included in the present disclosure will be described in further detail below.

[0011] The present disclosure encompasses a pharmaceutical composition for treating or preventing hyperammonemia, comprising rifaximin and a conjugated bile acid. In this specification, the pharmaceutical composition for treating or preventing hyperammonemia may be referred to as the "pharmaceutical composition for treating or preventing hyperammonemia of the present disclosure."

[0012] Rifaximin (2S,16Z,18E,20S,21S,22R,23R,24R,25S,26R,27S,28E)-5,6,21,23-Tetrahydroxy-27-methoxy-2,4,11,16,20,22,24,26-octamethyl-1,15-dioxo-1,2-dihydro-2,7-(epoxypentadeca-[1,11,13]trienoimino)furo[2",3":7',8']naphtho-[1',2':4,5]imidazo[1,2-a ]pyridin-25-yl acetate) is a rifamycin antibiotic known to exhibit antibacterial activity against aerobic gram-positive bacteria and facultative anaerobic gram-negative bacteria. Rifaximin is already used as a treatment for hyperammonemia in hepatic encephalopathy.

[0013] Rifaximin used in the present disclosure may be amorphous or crystalline, and the crystalline form of rifaximin is not limited to a specific crystalline form, and various polymorphs (e.g., crystalline polymorphs α, β, and γ) may be used.

[0014] Rifaximin can be produced by conventional methods, for example, Patent Document 1, JP-A No. 2007-509904, JP-A No. 2008-531623, etc.

[0015] Examples of bile acids constituting the conjugated bile acids used in the present disclosure include primary bile acids and secondary bile acids. Examples of primary bile acids include cholic acid and chenodeoxycholic acid. Examples of secondary bile acids include deoxycholic acid and lithocholic acid. In the conjugated bile acids used in the present disclosure, compounds that conjugate with bile acids include, for example, amino acids such as glycine and taurine, with taurine being preferred. Examples of conjugated bile acids include taurodeoxycholic acid, glycodeoxycholic acid, etc. Among these, taurodeoxycholic acid is preferred. The conjugated bile acid may be in the form of a salt. Specific examples include pharmaceutically acceptable salts such as salts with alkali metals such as potassium and sodium, and salts with alkaline earth metals such as calcium and magnesium. The conjugated bile acid may also be in the form of a solvate (e.g., hydrate, alcoholate, etc.). The conjugated bile acid that can be used is not particularly limited, and may be one purified from bile, one chemically synthesized, or one that is commercially available. The conjugated bile acids can be used singly or in combination of two or more.

[0016] In the pharmaceutical composition for treating or preventing hyperammonemia of the present disclosure, the mass ratio of rifaximin to conjugated bile acids is not particularly limited and can be appropriately set, for example, at about 1:99 to 99:1.

[0017] The content of rifaximin in the pharmaceutical composition for treating or preventing hyperammonemia of the present disclosure is not particularly limited and can be set appropriately, for example, to about 1 to 99% by mass.

[0018] The content of conjugated bile acids in the pharmaceutical composition for treating or preventing hyperammonemia of the present disclosure is not particularly limited and can be set appropriately, for example, to about 1 to 99% by mass.

[0019] The pharmaceutical composition for treating or preventing hyperammonemia of the present disclosure comprises rifaximin and conjugated bile acids, and may further comprise other ingredients. Examples of such other ingredients include pharmaceutically acceptable bases, carriers, and / or additives (e.g., solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, antioxidants, preservatives, coating agents, colorants, and other agents such as gastric mucosa protectants). These ingredients may be used singly or in combination of two or more.

[0020] The dosage form of the pharmaceutical composition for treating or preventing hyperammonemia of the present disclosure is not particularly limited, and examples include tablets, pills, capsules, powders, fine granules, granules, liquids, troches, jellies, injections, plasters, extracts, suppositories, suspensions, tinctures, ointments, poultices, nasal drops, inhalants, liniments, lotions, aerosols, etc. Among these, tablets are preferred.

[0021] The pharmaceutical composition for treating or preventing hyperammonemia of the present disclosure can be prepared by combining rifaximin and conjugated bile acids, and, if necessary, other ingredients, in a conventional manner.

[0022] As shown in the Examples below, the pharmaceutical composition for treating or preventing hyperammonemia of the present disclosure can enhance the rifaximin sensitivity of Ruminococcus gnavus and Streptococcus salivarius, causative bacteria of hyperammonemia, by conjugating bile acids. Therefore, the pharmaceutical composition can be suitably used for treating or preventing hyperammonemia. Examples of hyperammonemia include those caused by viral hepatitis, alcoholic hepatitis, non-alcoholic steatohepatitis, autoimmune hepatitis, and liver cirrhosis. Furthermore, the pharmaceutical composition for treating or preventing hyperammonemia of the present disclosure is capable of increasing the rifaximin sensitivity of Ruminococcus gnavus and Streptococcus salivarius, which are causative bacteria of hyperammonemia, by using conjugated bile acids, and is therefore expected to be effective even for patients for whom rifaximin administration does not significantly improve the efficacy.

[0023] The dosage (intake) of the pharmaceutical composition for treating or preventing hyperammonemia of the present disclosure is not particularly limited and is determined based on the age, weight, sex, severity of symptoms, administration method, etc. of the subject to be treated. For example, the dosage of the pharmaceutical composition for treating or preventing hyperammonemia of the present disclosure can be about 1 to 2,000 mg per day as the dosage of rifaximin. This dosage may be, for example, about 10 to 2,000 mg, or about 100 to 1,500 mg. Furthermore, the dosage of the pharmaceutical composition for treating or preventing hyperammonemia of the present disclosure can be about 1 to 1,000 mg per day as the dosage of conjugated bile acids. This dosage may be, for example, about 10 to 1,000 mg, or about 100 to 1,000 mg. The pharmaceutical composition for treating or preventing hyperammonemia of the present disclosure may be administered once a day, or may be administered in divided doses (for example, 2, 3, 4, 5 times, etc.) per day.

[0024] The subject of administration of the pharmaceutical composition for treating or preventing hyperammonemia of the present disclosure is preferably a mammal. It may be not only a human but also a non-human mammal. Examples of the human subject include hyperammonemia patients, humans suspected of having hyperammonemia, hepatic encephalopathy patients, humans suspected of having hepatic encephalopathy, humans receiving rifaximin, and humans who have received rifaximin. Examples of non-human mammals include mammals kept as pets, livestock, laboratory animals, etc. Examples of such non-human mammals include dogs, cats, monkeys, cows, horses, sheep, goats, pigs, rabbits, mice, rats, camels, and llamas.

[0025] Methods for administering the pharmaceutical composition for treating or preventing hyperammonemia according to the present disclosure include, for example, oral administration and parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous, peritoneal, rectal, transdermal, topical, etc.) administration, etc. Among these, oral administration is preferred.

[0026] The present disclosure also encompasses a pharmaceutical composition for treating or preventing hepatic encephalopathy, comprising rifaximin and a conjugated bile acid. In this specification, the pharmaceutical composition for treating or preventing hepatic encephalopathy may be referred to as the "pharmaceutical composition for treating or preventing hepatic encephalopathy of the present disclosure."

[0027] The above description of the pharmaceutical composition for treating or preventing hyperammonemia of the present disclosure can be applied to the pharmaceutical composition for treating or preventing hepatic encephalopathy of the present disclosure.

[0028] As shown in the Examples below, the pharmaceutical composition for treating or preventing hepatic encephalopathy of the present disclosure can enhance the rifaximin sensitivity of Ruminococcus gnavus and Streptococcus salivarius, causative bacteria of hyperammonemia, by conjugated bile acids. Therefore, the composition can be suitably used for treating or preventing diseases caused by hyperammonemia. Examples of diseases caused by hyperammonemia include hepatic encephalopathy. It may also be possible to enhance the effect on rifaximin-susceptible bacteria other than Ruminococcus gnavus and Streptococcus salivarius. For example, it may be applicable to bacteria that exacerbate enteritis. Examples of rifaximin-susceptible bacteria other than Ruminococcus gnavus and Streptococcus salivarius include the genera Streptococcus, Clostridium, Proteus, and Bacteroides. Furthermore, the pharmaceutical composition for treating or preventing hepatic encephalopathy of the present disclosure is capable of increasing the rifaximin sensitivity of Ruminococcus gnavus and Streptococcus salivarius, which are causative bacteria of hyperammonemia, by using conjugated bile acids, and is therefore expected to be effective even for patients for whom rifaximin administration does not significantly improve the efficacy.

[0029] The present disclosure also encompasses a pharmaceutical composition comprising a conjugated bile acid, the pharmaceutical composition being for use in a human being to be or who has been administered rifaximin. In this specification, the pharmaceutical composition may be referred to as the "pharmaceutical composition of the present disclosure."

[0030] For the conjugated bile acids contained in the pharmaceutical composition of the present disclosure, the above description of the pharmaceutical composition for treating or preventing hyperammonemia of the present disclosure can be cited.

[0031] The content of conjugated bile acids in the pharmaceutical composition of the present disclosure is not particularly limited and can be set appropriately, for example, to about 1 to 99% by mass.

[0032] The pharmaceutical composition of the present disclosure contains a conjugated bile acid and may further contain other ingredients, which may be the same as those described above for the pharmaceutical composition for treating or preventing hyperammonemia of the present disclosure.

[0033] The dosage form and preparation method of the pharmaceutical composition of the present disclosure can be referenced from the above description of the pharmaceutical composition for treating or preventing hyperammonemia of the present disclosure.

[0034] As shown in the Examples below, the pharmaceutical composition of the present disclosure can enhance the rifaximin sensitivity of Ruminococcus gnavus and Streptococcus salivarius, causative bacteria of hyperammonemia, by conjugating bile acids. Therefore, the pharmaceutical composition is suitable as a pharmaceutical composition for use in humans to which or who have been administered rifaximin (as an active ingredient). Because rifaximin is used as a therapeutic agent for hyperammonemia, the pharmaceutical composition of the present disclosure can be suitably used for the treatment or prevention of hyperammonemia. The pharmaceutical composition of the present disclosure can also be used for the treatment or prevention of diseases caused by hyperammonemia. Examples of diseases caused by hyperammonemia include hepatic encephalopathy. Furthermore, the pharmaceutical composition of the present disclosure is capable of increasing the rifaximin sensitivity of Ruminococcus gnavus and Streptococcus salivarius, which are causative bacteria of hyperammonemia, by using conjugated bile acids, and is therefore expected to be effective even for patients for whom rifaximin administration does not significantly improve.

[0035] The dosage (intake) of the pharmaceutical composition of the present disclosure is not particularly limited and is determined depending on the age, weight, sex, severity of symptoms, administration method, etc. of the subject to be administered. For example, the dosage of conjugated bile acids can be about 1 to 1,000 mg per day. The dosage may be, for example, about 10 to 1,000 mg, or about 100 to 1,000 mg. The pharmaceutical composition of the present disclosure may be administered once a day, or may be administered in divided doses (for example, 2, 3, 4, 5, etc.) per day.

[0036] The subjects and administration methods of the pharmaceutical composition of the present disclosure can be referenced from the above description of the pharmaceutical composition for treating or preventing hyperammonemia of the present disclosure.

[0037] The present disclosure also encompasses an enhancer for the therapeutic or preventive effect of rifaximin on hyperammonemia or hepatic encephalopathy, which comprises a conjugated bile acid. In this specification, such a pharmaceutical composition may be referred to as the "enhancement agent of the present disclosure."

[0038] For the enhancer of the present disclosure, the above description of the pharmaceutical composition of the present disclosure can be cited.

[0039] As shown in the Examples below, the enhancer of the present disclosure can enhance the rifaximin sensitivity of Ruminococcus gnavus and Streptococcus salivarius, which are causative bacteria of hyperammonemia, by conjugating bile acids, making it suitable as an enhancer for the therapeutic or preventive effect of rifaximin on hyperammonemia or hepatic encephalopathy. Furthermore, the enhancer of the present disclosure can enhance the rifaximin sensitivity of Ruminococcus gnavus and Streptococcus salivarius, which are causative bacteria of hyperammonemia, by using conjugated bile acids, and is therefore expected to be effective even in patients for whom rifaximin administration does not significantly improve the response.

[0040] In this specification, the term "comprising" includes "consisting essentially of" and "consisting of." Furthermore, the present disclosure encompasses all arbitrary combinations of the constituent elements described in this specification.

[0041] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to specify the subject matter encompassed by the present disclosure, i.e., the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein. [Example]

[0042] The contents of the present disclosure will be specifically explained using the following experimental examples. However, the present disclosure is not limited to these examples. In the following, unless otherwise specified, experiments were performed under atmospheric pressure and room temperature conditions. Furthermore, unless otherwise specified, "%" means "% by weight."

[0043] Clinical samples Between April 2017 and March 2020, patients with liver cirrhosis who visited Osaka City University Hospital and developed grade I or II hepatic encephalopathy according to the West Haven Criteria and hyperammonemia with a peripheral venous blood ammonia concentration of 70 μg / dL or higher were included. The fecal intestinal flora of 28 patients (HE: Hepatic Encephalopathy) was examined, who did not meet the following exclusion criteria (hematemesis, severe infection, acute hepatitis, acute liver failure, or acute exacerbation of chronic hepatitis). Rifaximin (RFX), a poorly absorbed antibiotic, is used to treat patients with hepatic encephalopathy caused by hyperammonemia because it can eliminate ammonia-producing bacteria in the intestine. Of 28 patients with hepatic encephalopathy, 20 were administered rifaximin, and stool samples were collected from 9 of these patients 4 weeks after RFX administration, allowing for intestinal microbiota analysis. "Responders" were defined as patients whose plasma ammonia levels decreased by 25% or more with RFX, and "non-responders" were defined as patients whose plasma ammonia levels decreased by less than 25% or increased with RFX. Stool samples from healthy individuals were collected from participants who visited the Medicity21 Health Check Center at Osaka City University Hospital between January 2020 and June 2020. A total of 73 participants were excluded based on the following four exclusion criteria: (a) age <50 years; (b) BMI between 18 and 25 kg / m 2 (c) Subjects with a history of liver disease and diabetes; (d) Subjects who had used antibiotics and probiotics, including RFX, within 2 weeks prior to sampling. After exclusion, the remaining 26 subjects were designated as healthy controls (HC). Information about each sample is shown in Table 1. This study was conducted with approval from the Ethics Committee of the Osaka City University Graduate School of Medicine (approval number: 3722). The study was conducted in accordance with the Declaration of Helsinki II, and written informed consent was obtained from all participants.

[0044] [Table 1]

[0045] Human stool sample collection and DNA extraction methods Human fecal samples were collected using a stool sampling kit (Techno Suruga Lab, brush type). Fecal DNA extraction was outsourced to Biken Biomics. Fecal DNA was extracted using an automated DNA extractor (GENE PREP STAR PI-480, Kurabo Industries, Ltd.), and the concentration of the extracted DNA was measured using Qubit Assays (Thermo Fisher Scientific Inc., DE, USA).

[0046] Amplification, sequencing, and analysis of the 16S ribosomal RNA gene DNA library preparation and sequencing were outsourced to Biken Biomics. Each DNA library was created by amplifying the hypervariable V1-V2 region of bacterial 16S ribosomal RNA (rRNA) using the primer set 27Fmod: 5'-AGRGTTTGATCMTGGCTCAG-3' and 338R: 5'-TGCTGCCTCCCGTAGGAGT-3' according to the Illumina 16S Metagenomic Sequencing Library Preparation Guide. 251-bp paired-end sequencing of the amplicons was performed on a MiSeq system (Illumina) using the MiSeq Reagent v2 500-cycle kit. The sequences obtained with the Illumina MiSeq were analyzed using the Quantitative Insights into Microbial Ecology 2 version 2019.4 (QIIME2) pipeline. The resulting paired-end sequences were denoised and analyzed using the DADA2 R library in QIIME2. The obtained DNA sequence information was then used to classify ASVs with 99% homology using the SILVA 132 16S rRNA gene sequence database. Comparative bacterial abundance analysis was performed using the linear discriminant analysis (LDA) effect size (LEfSe) online galaxy version. The results of the QIIME2 and LEfSe analyses are shown in Figures 2A and 2B. The genera prevalent in the HE group are shown in Figure 2C. A graph showing the relative abundance of bacteria at the species level is shown in Figure 2D.

[0047] Comparing the individual bacterial flora compositions, we found that the genus Streptococcus was more prevalent in the HE group (Fig. 2A). The genera significantly prevalent in the HE group were Lactobacillus, Streptococcus, Bifidobacterium, and Veillonella (Fig. 2B, C). The most prevalent bacterial species in the HE group was Streptococcus salivarius (Fig. 2D).

[0048] Bacterial quantitative PCR and analysis Bacterial DNA was isolated from mouse feces using a kit (QIAamp Fast DNA Stool Mini Kit, Qiagen). DNA was amplified and quantified using SYBR Premix EX Taq II (Takara, Shiga, Japan) with bacterial-specific PCR primers on a StepOnePlus real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). The abundance of Streptococcus salivarius in human fecal DNA (quantified by 16S rRNA and UreC (the gene encoding the central enzyme in the urease operon)) assessed by quantitative PCR (mean ± SEM, Mann-Whitney U test) is shown in Figure 3A. The correlation between the abundance of S. salivarius 16S rRNA gene DNA and UreC DNA based on the correlation coefficient is shown in Figure 3B.

[0049] There was a significant correlation between the abundance of S. salivarius 16S rRNA gene DNA and UreC DNA, indicating that most of the S. salivarius in the samples from the HE group used in the test were bacteria that possessed UreC.

[0050] Urease test (measurement of ammonia concentration, which reflects urease activity) Urease activity was measured using Streptococcus salivarius NCTC 7366 strain (sometimes referred to as Ur+SS 7366) and Streptococcus salivarius ATCC 25975 strain (sometimes referred to as Ur-SS 25975). LabAssay™ Ammonia (FUJIFILM Wako Pure Chemical Industries, Ltd.) was used to measure the ammonia concentration in the reaction solution for urease activity measurement. The reaction solution was allowed to turn red, and the value measured using an absorbance meter (wavelength OD545) was used. The results are shown in Figure 3C.

[0051] It was found that strain 7366 has urease activity, but strain 25975 does not.

[0052] experimental animals Eight-week-old female C57BL / 6N mice were purchased from CLEA Japan. All animal experiments were approved by the Animal Care and Use Committee (approval numbers: 17206, 18079).

[0053] Mouse experiments Experimental liver fibrosis was induced in mice by administering carbon tetrachloride (CCl4) twice weekly. Eight-week-old female C57BL / 6N mice were intraperitoneally (ip) injected with 0.5 mg / kg body weight of CCl4 (diluted 20% in olive oil) twice weekly for 16 weeks. Control mice were injected with olive oil alone. To eliminate intestinal flora, mice were given water containing 1 g / L ampicillin, 1 g / L neomycin, 0.5 g / L metronidazole, and 0.5 g / L vancomycin for 14 days from weeks 12 to 14. Metronidazole, which has no bactericidal effect on Streptococcus spp., was administered alone for an additional 3 days to suppress the growth of other bacteria. To confirm the importance of urease, two strains, S. salivarius ATCC 25975, which lacks urease activity, and S. salivarius NCTC 7366, which has urease activity, were used, and 5.0 × 10 9Mice were administered 200 μL of saline containing 100 CFU of bacteria daily for 14 days between 14 and 16 weeks of age using a plastic feeding tube (Fuchigami).

[0054] Measurement of plasma ammonia in mice with liver fibrosis induced by intraperitoneal injection of carbon tetrachloride Mouse blood samples were collected from the right ventricle and centrifuged at low speed to extract plasma. Plasma ammonia was measured immediately after centrifugation using a FUJIFILM DRI-CHEM NX10N slide and a DRI-CHEM NH3-PII slide (FUJIFILM). The results (mean ± SEM, one-way analysis of variance) are shown in Figure 4A.

[0055] It was confirmed that plasma ammonia levels were significantly higher in mice administered with the urease-active 7366 strain.

[0056] Bacterial genome isolation and species identification Bacterial DNA was extracted using NucleoSpin™ Microbial DNA (TaKaRa). To identify the isolated bacterial species, the 16S rRNA gene was amplified from the bacterial DNA using the bacterial universal PCR primers Bact-27F and Bact-1492R, and the 16S rRNA gene sequence of the PCR product was determined. Nucleotide sequences were determined using an ABI 3130x1 capillary sequencer (Applied Biosystems) with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA) and sequencing primers (Bact-27F, Bact-1492R). The resulting sequences were analyzed using the NCBI (National Center for Biotechnology Information) BLAST and the 16S ribosomal RNA sequence database for species identification (identification criteria: identity > 97%, coverage > 95%).

[0057] Bacterial quantitative PCR and analysis DNA isolated from mouse feces was amplified and quantified using bacterial-specific PCR primers using SYBR Premix EX Taq II (Takara, Shiga, Japan) and a StepOnePlus real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). The abundance of S. salivarius in mouse fecal DNA samples quantified by quantitative PCR of 16S rRNA (mean ± SEM, Mann-Whitney U test) and the abundance of UreC (a subunit of the urease enzyme) are shown in Figure 4B.

[0058] When mice were administered S. salivarius, the bacteria were present in the feces of the mice, and UreC was detected in the group administered the 7366 strain.

[0059] Bacterial culture Streptococcus salivarius NCTC7366 was cultured anaerobically overnight at 37°C in brain heart infusion (BHI) medium (Nippon Suisan Honsha). Ruminococcus gnavus ATCC 29149 was cultured anaerobically overnight at 37°C in modified Gifu anaerobic medium (mGAM) (Nippon Suisan Honsha).

[0060] Bacterial growth assay Bacterial growth was analyzed by measuring colorimetric values ​​at 595 nm (OD595) using a microplate reader (Bio-Rad, Hercules, CA). To examine bacterial susceptibility to RFX, strains grown overnight in mGAM medium were adjusted to an OD595 of 0.01 and plated in 2 wells onto a 96-well microtiter plate. -11 From 2 7 Rifaximin was added in the range of μg / mL, and the mixture was incubated at 37°C for 24 hours, after which the OD595 value was measured. The abundance of S. salivarius in the presence of rifaximin (using seven concentrations of serial two-fold dilutions starting from 0.125 μg / ml) in BHI medium is shown in FIG. 5 (mean ± SD).

[0061] S. salivarius was confirmed to be susceptible to rifaximin.

[0062] We investigated the changes in plasma ammonia levels before and 4 weeks after RFX administration in two groups, responders and non-responders (responders: n = 5, non-responders: n = 4). The results are shown in Figure 6(A).

[0063] After RFX administration, plasma ammonia levels were confirmed to decrease in responders.

[0064] The total amount of fecal bacteria in patients in the two groups (responders and non-responders) was measured by quantitative PCR of the 16S rRNA gene before and 4 weeks after RFX administration. The results are shown in Figure 6(B).

[0065] It was confirmed that there was no significant difference in the amount of intestinal bacteria itself.

[0066] The relative abundance of S. salivarius (compared by 16S rRNA gene abundance determined by Qiime2 analysis, Wilcoxon signed-rank test) was measured before and 4 weeks after RFX administration in the two groups of responders and non-responders. The results are shown in Figure 6(C).

[0067] It was confirmed that S. salivarius was reduced by administration of rifaximin.

[0068] The amount of S. salivarius was measured by UreC quantitative PCR in feces from patients in the two groups (responders and non-responders) before and 4 weeks after RFX administration. The results (paired t-test) are shown in Figure 7.

[0069] A significant reduction in S. salivarius was confirmed, especially in responders.

[0070] These findings suggest that S. salivarius is one of the causative bacteria of hyperammonemia in responders.

[0071] Four weeks after RFX administration, the abundance of bacteria was compared between responders and non-responders by LEfSe analysis. The results of analyzing the intestinal bacterial species that were abundant in non-responders are shown in Figure 8A.

[0072] The only enterobacterial species that was prevalent in non-responders was Ruminococcus gnavus (LDA score > 3).

[0073] The relative abundance of R. gnavus (quantified from Qiime2 analysis data) was measured before and 4 weeks after RFX administration, and the results are shown in Figure 8B.

[0074] In non-responders, administration of RFX did not reduce R. gnavus.

[0075] Urease activity was measured using R. gnavus. Recombinant urease (rUrease) was used as a control. The results are shown in Figure 8C.

[0076] It was found that R. gnavus does not have urease activity.

[0077] Determination of in vitro urease activity of urease-producing bacteria. Bacteria were pre-cultured at 37°C for 24 hours, and 1 ml of the pre-culture solution was added to 50 ml of medium and incubated at 37°C for 18 hours. The culture medium was centrifuged at 3,000 × g for 10 minutes at 4°C, and the pellet was washed twice with 0.85% NaCl to remove medium components, followed by a urease test.

[0078] Bacterial culture Streptococcus salivarius NCTC 7366 was cultured anaerobically overnight at 37°C in brain heart infusion (BHI) medium (Nippon Suisan Honsha). Ruminococcus gnavus ATCC29149 was cultured anaerobically overnight at 37°C in modified Gifu anaerobic medium (mGAM) (Nippon Suisan Honsha). Citrobacter freundii (CF) and Lactobacillus salivarius (LS) were isolated from patient fecal samples and cultured anaerobically at 37°C on mGAM agar medium. Streptococcus salivarius or Citrobacter freundii was resuspended in buffer and incubated with Ruminococcus gnavus or Lactobacillus salivarius (final concentrations of 500 mg / L NaCl, 200 mg / L KH2PO4, 200 mg / L urea, and 30 mg / L phenol red). After 24 hours of shaking at 37°C, the color development was measured spectrophotometrically at OD545. The results are shown in Figure 9A. Data were expressed as mean ± SD. One-way analysis of variance was applied to each group with correction for multiple comparisons: 1: control, RG, and LS; 2: control, RG, and LS vs. SS; 3: control, RG, and LS vs. CF. LS (Lactobacillus salivarius) was used as a negative control, as it did not enhance urease activity by R. gnavus.

[0079] As shown in Figure 9A, co-cultivation of R. gnavus with urease-containing bacteria (SS; Streptococcus salivarius, CF; Citrobacter freundii) increased urease activity.

[0080] R. gnavus was cultured with recombinant urease (five serial dilutions, starting from 1 unit, starting with a two-fold dilution), and urease activity was measured. The results (mean ± SD, Student's t-test) are shown in Figure 9B (RG+). The results of measuring urease activity using recombinant urease alone (five serial dilutions, starting from 1 unit, starting with a two-fold dilution) are also shown (RG-).

[0081] It was confirmed that the combined use of R. gnavus and recombinant urease increased the urease activity of the recombinant urease.

[0082] These findings suggest that R. gnavus is one of the causative bacteria of hyperammonemia in non-responders, and that R. gnavus enhances the urease activity of other ammonia-producing bacteria.

[0083] Quantitation of bile acids in human plasma by LC-MS / MS Human plasma metabolites were extracted from 50 μL of a suspension in 250 μL of 50% methanol in Milli-Q water containing 20 μM CSA (10-camphorsulfonic acid) as an internal standard. Liquid chromatography (LC) separation was performed on an Agilent 1290 UPLC system (Agilent Technologies) with gradient elution on an ACQUITY UPLC HSS T3 column (1.8 μm, 50 mm x 2.1 mm ID; Waters). Mass spectrometry (MS) analysis was performed on an Agilent 6490 triple quadrupole mass spectrometer (Agilent Technologies).

[0084] Figure 10A shows a heat map of plasma bile concentrations in responder patients (R) and non-responder patients (NR) before RFX administration. FIG. 10B shows the amounts of CA (cholic acid), DCA (deoxycholic acid), TDCA (taurodeoxycholic acid), and GDCA (glycodeoxycholic acid) (mean±SEM, Mann-Whitney U test).

[0085] The results showed that the conjugated secondary bile acids TDCA (taurodeoxycholic acid) and GDCA (glycodeoxycholic acid) were low in non-responders and significantly higher in responders.

[0086] Bacterial growth assay Bacterial growth was analyzed by measuring colorimetric values ​​at 595 nm (OD595) using a microplate reader (Bio-Rad, Hercules, CA). To test the susceptibility of bacteria to RFX, strains grown overnight in mGAM medium were adjusted to an OD595 of 0.01 and plated in 2 wells on a 96-well microtiter plate. -11 From 2 7 After incubation at 37°C for 24 hours with rifaximin at concentrations ranging from 0.1 to 1 μg / mL, the strains were grown overnight in mGAM and adjusted to an OD of 0.1. The OD of the strains was then measured. To examine whether bile acids increase the susceptibility of bacteria to RFX, the strains were grown overnight in mGAM and adjusted to an OD of 0.1. The OD of the strains was then measured at 2 μg / mL with rifaximin and TDCA (5 mM for R. gnavus, 1 mM for S. salivarius). -12 to 2 -3 The mGAM medium was added to a concentration in the range of μg / mL, and the OD595 value was measured in the same manner. The abundance of R. gnavus (left) and S. salivarius (right) in the presence of RFX (seven two-fold serial dilutions starting from 0.016 μg / ml) with or without TDCA (5 mM for R. gnavus, 1 mM for S. salivarius) is shown in Figure 11 (mean ± SD, Student's t-test). The abundance of bacteria was measured by absorbance at OD595.

[0087] The rifaximin sensitivity of S. salivarius and R. gnavus was found to be higher in the presence of taurodeoxycholic acid than in the absence of taurodeoxycholic acid. In other words, the use of taurodeoxycholic acid increased the rifaximin sensitivity of S. salivarius and R. gnavus, which are causative bacteria of hyperammonemia.

[0088] Bacterial growth assays were performed in the same manner using DCA instead of TDCA. The abundance of each strain in the absence of RFX is shown in Figure 12, and the abundance of each strain in the presence of RFX is shown in Figure 13.

[0089] The addition of unconjugated deoxycholic acid did not confirm the sensitivity-enhancing effect of rifaximin.

Claims

1. A pharmaceutical composition for treating or preventing hyperammonemia, comprising rifaximin and a conjugated bile acid, wherein the conjugated bile acid is taurodeoxycholic acid.

2. A pharmaceutical composition for treating or preventing hepatic encephalopathy, comprising rifaximin and a conjugated bile acid, wherein the conjugated bile acid is taurodeoxycholic acid.

3. Conjugated bile acids, including an agent for enhancing the therapeutic or preventive effect of rifaximin on hyperammonemia or hepatic encephalopathy; Here, the conjugated bile acid is taurodeoxycholic acid.

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