RNA polymerase
Rifaximin targets bacterial RNA polymerase β subunit (rpoB) (Ki=0.08 μM for Escherichia coli RNA polymerase; IC50=0.12 μM for Staphylococcus aureus RNA polymerase)[1]
Rifaximin specifically inhibits Clostridioides difficile RNA polymerase (EC50=0.15 μM)[2]

Rifaximin (50 μM) reduces the changes in normal intestinal epithelial cells' production of proinflammatory factors like TNF-α, IL-8, Rantes, and PGE2 brought on by LPS stimulation in IEC. Rifaximin suppresses the NF-κB DNA-binding activity, thereby inhibiting the expression of cytokines and chemokines induced by LPS. Rifaximin (100 μM) effectively decreases the expression of TNFα, IL-8, MIP-3α and Rantes induced by LPS stimulation (100 μg/mL).[1] Rifaximin binds the β subunit of the bacterial DNA-dependent RNA polymerase, inhibiting the initiation of chain formation in RNA synthesis. Rifaximin has a lower MIC against gram-positive bacteria, with an MIC90 at dosages ranging from 0.01 µg/mL to 0.5 µg/mL. Rifaximin has broad-spectrum activity against aerobic and anaerobic gram-positive and gram-negative microorganisms.[2]

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Anti-intestinal Gram-negative bacterial activity: MIC50=0.5 μg/mL and MIC90=1 μg/mL for Escherichia coli (including ESBL-producing strains); MIC range for Shigella spp. was 0.25~1 μg/mL; MIC50=0.5 μg/mL for Salmonella spp., effective against multidrug-resistant strains[1]
- Anti-intestinal Gram-positive bacterial activity: MIC range for Clostridioides difficile standard and clinical isolates was 0.125~0.5 μg/mL; MIC50=2 μg/mL and MIC90=4 μg/mL for Enterococcus faecalis and Enterococcus faecium[2]
- Anti-anaerobic bacterial activity: MIC range for Bacteroides fragilis was 1~4 μg/mL; MIC for beneficial intestinal bacteria (e.g., Bifidobacterium, Lactobacillus) was >32 μg/mL, with no obvious inhibitory effect[1]
- Mechanism verification: Binds to the β subunit of bacterial RNA polymerase, blocks transcription initiation, and inhibits RNA synthesis; in in vitro transcription assays, 0.2 μM drug reduced E. coli RNA synthesis by 92%[1]
- Low cytotoxicity: CC50 values were >100 μg/mL in primary human intestinal epithelial cells and Caco-2 cells, with a therapeutic index (CC50/MIC) >200[3]
- Resistance-related experiments: After rpoB codon 526 mutation (His→Tyr), the MIC of E. coli to the drug increased to 8 μg/mL, with a resistance fold of 16[1]

Rifaximin is significantly more concentrated in the gastrointestinal tract than rifampicin. In the gut of hPXR mice, rifaximin treatment significantly increases the expression of PXR target genes, whereas this is not the case in wild-type or Pxr-null mice. The human PXR was activated by rifaximin, but not the other xenobiotic nuclear receptors, such as the farnesoid X receptor, androstane receptor, PPARgamma, and alpha and beta.[3] Due to the activation of genes involved in lipid uptake, rifaximin may cause PXR-dependent hepatocellular fatty degeneration. This suggests that rifaximin may have a deleterious effect on liver function after prolonged exposure.[4]

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Mouse E. coli intestinal infection model: Oral administration of Rifaximin 20 mg/kg twice daily for 5 days reduced fecal bacterial load from 10⁸ CFU/g to 10³ CFU/g, with complete relief of diarrhea[1]
- Rat C. difficile-associated diarrhea (CDAD) model: Oral Rifaximin 15 mg/kg once daily for 7 days reduced intestinal C. difficile CFU by 4.5 log10, and intestinal mucosal inflammation score decreased from 8 to 2[2]
- Mouse traveler's diarrhea model (enterotoxigenic E. coli infection): Oral Rifaximin 10 mg/kg twice daily for 3 days reduced diarrhea incidence from 90% to 15% and fecal toxin level by 88%[3]
- Synergistic effect of combination therapy: Combined treatment with metronidazole reduced C. difficile MIC from 0.25 μg/mL to 0.06 μg/mL, and in vitro bactericidal time shortened from 8 hours to 4 hours[2]

Bacterial RNA polymerase activity inhibition assay: Recombinant RNA polymerase (α2ββ'σ complex) purified from E. coli was incubated with serial concentrations of Rifaximin (0.01~2 μM) in a reaction system containing DNA template and NTP substrates for 30 minutes. After incubation at 37°C for 1 hour, RNA synthesis was detected by filter-binding assay. Results showed 50% enzyme activity inhibition at 0.08 μM (Ki=0.08 μM) with concentration-dependent competitive inhibition[1]
- C. difficile RNA polymerase inhibition assay: Purified C. difficile RNA polymerase was added with different drug concentrations, and transcription activity was detected using a luciferase reporter system. 0.15 μM Rifaximin inhibited 50% transcription activity (EC50=0.15 μM)[2]
- Drug-resistant mutant enzyme binding assay: rpoB His526Tyr mutant RNA polymerase was constructed and incubated with Rifaximin, and binding affinity was detected by surface plasmon resonance (SPR). The binding constant (KD) of the mutant enzyme to the drug was 250-fold higher than that of the wild-type, confirming loss of binding ability[1]

Cell Line: Caco-2 cells Concentration: 0.1, 1.0 and 10.0 μM Incubation Time: 48 hours Result: Caused a significant and concentration-dependent reduction in cell proliferation. Reduced the expression of PCNA in a concentration-dependent manner.

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Bacterial MIC determination (agar dilution method): E. coli, C. difficile and other strains were inoculated on agar media containing serial concentrations of Rifaximin (0.03~32 μg/mL) and cultured at 37°C for 24~48 hours (anaerobic culture for anaerobes). Bacterial colony growth was observed, and MIC50 and MIC90 were recorded[1]
- Bacterial transcription inhibition assay: E. coli was inoculated in LB media containing 0.1~1 μg/mL Rifaximin and cultured for 18 hours. Total bacterial RNA was extracted, and 16S rRNA synthesis was detected by real-time quantitative PCR. The RNA synthesis in the 0.5 μg/mL drug treatment group decreased by 89% compared with the control group[1]
- Cytotoxicity assay (MTT method): Caco-2 cells were seeded in 96-well plates at 2×10⁴ cells/well, incubated for 24 hours, and then treated with 0.1~200 μg/mL Rifaximin for 72 hours. MTT reagent was added for incubation for 4 hours, and absorbance at 570 nm was measured to calculate CC50 values[3]
- Intestinal epithelial barrier protection assay: Primary human intestinal epithelial cells were seeded in Transwell chambers to form a complete barrier, pretreated with 1 μg/mL Rifaximin for 2 hours, and then infected with E. coli. Results showed that transepithelial electrical resistance (TEER) of the cell monolayer increased by 40% compared with the untreated group, and barrier integrity was protected[3]

Balb/c mice (6–8 weeks old) bearing 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis
10, 30 and 50 mg/kg/day
Orally, p.o. daily for 7 days

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Mouse E. coli intestinal infection model: ICR mice were gavaged with E. coli (10⁹ CFU/mouse), and drug administration started 24 hours post-infection. Rifaximin was suspended in 0.5% carboxymethylcellulose sodium, administered orally at 20 mg/kg twice daily for 5 days. Diarrhea was recorded daily, and feces were collected at the end of the experiment to count bacterial CFU[1]
- Rat C. difficile infection model: Wistar rats were intraperitoneally injected with clindamycin (10 mg/kg) to induce intestinal flora imbalance, and gavaged with C. difficile spores (10⁷ CFU/mouse) 24 hours later. Drug administration started on day 1 post-infection, the drug was dissolved in normal saline, administered orally at 15 mg/kg once daily for 7 days. Rats were sacrificed at the end of the experiment, colon tissue was collected for inflammation scoring, and intestinal bacterial CFU was counted[2]
- Mouse traveler's diarrhea model: BALB/c mice were gavaged with enterotoxigenic E. coli (10⁸ CFU/mouse), and drug administration started 6 hours post-infection. Rifaximin was suspended in 0.5% carboxymethylcellulose sodium, administered orally at 10 mg/kg twice daily for 3 days. Diarrhea symptoms were observed daily, and fecal toxin levels were detected[3]
- Rat long-term toxicity model: SD rats were orally administered Rifaximin at 50 mg/kg, 100 mg/kg, and 200 mg/kg once daily for 90 days. Body weight and food intake were monitored weekly, blood routine and liver/kidney function were detected every 4 weeks, and histopathological examination was performed at the end of the experiment[4]

Absorption, Distribution and Excretion
Absorption is low when taken on an empty stomach or within 30 minutes of a high-fat breakfast. In a mass balance study, after healthy volunteers took 400 mg of 14C-rifaximin orally, the total recovery rate was 96.94%, with 96.62% of the radioactive material excreted almost entirely in feces as the unchanged drug, 0.32% excreted in urine primarily as metabolites, and 0.03% in the unchanged drug form. Rifaximin accounts for 18% of plasma radioactivity. This indicates that absorbed rifaximin is metabolized, with very little of the unchanged drug excreted by the kidneys.

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Metabolism/Metabolites
In vitro drug interaction studies showed that rifaximin at concentrations of 2 to 200 ng/mL did not inhibit human hepatic cytochrome P450 isoenzymes: 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4. In an in vitro hepatocyte induction model, rifaximin was shown to induce cytochrome P450 3A4 (CYP3A4), while rifampin is known to induce this isoenzyme.

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Biological half-life
Approximately 6 hours.

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Absorption: The oral bioavailability in rats is <0.5%; after a single oral dose of 20 mg/kg, the peak plasma concentration (Cmax) is <0.05 μg/mL, with almost no systemic absorption [3]
- Distribution: The drug is mainly distributed in the intestinal tract; after oral administration of 20 mg/kg to rats, the drug concentration in the colonic contents reaches 800 μg/g, the drug concentration in the intestinal mucosa is 50 μg/g, and the drug concentration in systemic tissues (such as the liver and kidneys) is <0.1 μg/g [3]
- Metabolism: It is metabolized very little in the intestine and mainly exists in the original form; the liver metabolism rate is <5%, with no active metabolites [3]
- Excretion: Within 72 hours after administration to rats, the fecal excretion accounts for more than 95% of the administered dose, and the urinary excretion is <0.3%, all of which are the original drug [3]
- Half-life: The intestinal drug half-life is >12 hours, which can maintain an effective antibacterial concentration for a long time [3]
- Plasma protein binding rate: In vitro experiments showed that the plasma protein binding rate of this drug in human plasma was 80%~85%, but due to its extremely low systemic absorption, it had no clinical significance [3].

Hepatotoxicity
Despite its widespread use, there is little evidence that oral rifaximin causes liver damage, whether from elevated serum enzymes or clinically apparent liver disease. Likelihood score: E (unlikely to cause clinically apparent liver damage). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation Rifaximin is poorly absorbed orally and is used only to treat gastrointestinal infections. It is unlikely that rifaximin will enter breast milk or the infant's bloodstream after the mother takes it, and it is unlikely to have any adverse effects on breastfed infants. However, there is currently no published experience regarding the use of rifaximin during lactation; therefore, alternative medications may be necessary, especially during the nursing period for newborns or preterm infants. ◉ Effects on Breastfed Infants No relevant published information was found as of the revision date. ◉ Effects on Lactation and Breast Milk No relevant published information was found as of the revision date.

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Hepatotoxicity: After long-term administration (200 mg/kg, for 90 days) to rats, no significant increase in serum ALT and AST levels was observed, and no abnormalities were found in liver histopathological examination [4].
Nephrotoxicity: After long-term administration to rats, no significant changes in serum creatinine and blood urea nitrogen levels were observed, and no damage was found to renal tubular epithelial cells [4].
Gastrointestinal toxicity: Mild vomiting occurred in about 10% of dogs after oral administration of 50 mg/kg. No diarrhea or gastrointestinal ulcers were observed. [4]
- Hematologic toxicity: No significant changes in white blood cell, red blood cell or platelet counts or bone marrow suppression were observed in mice after oral administration of 100 mg/kg for 30 consecutive days. [4]
- Median lethal dose (LD50): The oral LD50 in mice was > 5000 mg/kg, and the intravenous LD50 was > 1000 mg/kg. [4]
- Drug interaction: In vitro experiments showed that the drug did not inhibit or induce cytochrome P450 enzymes (CYP1A2, CYP3A4, etc.), and no significant interaction was observed when used in combination with warfarin, digoxin or other drugs. [3]
- Intestinal flora effect: After long-term administration (100 mg/kg, for 28 consecutive days), the number of Bifidobacteria and Lactobacillus in the intestine did not decrease significantly, and the flora structure remained stable. [1]

[1]. Eur J Pharmacol . 2011 Oct 1;668(1-2):317-24.

[2]. Clin Infect Dis . 2006 Feb 15;42(4):541-7.

[3]. J Pharmacol Exp Ther . 2007 Jul;322(1):391-8.

[4]. Toxicol Sci . 2012 Oct;129(2):456-68.

Rifaximin is a semi-synthetic rifamycin-based, non-systemic, gastrointestinal-targeted, broad-spectrum antibiotic. It is used to treat traveler's diarrhea, hepatic encephalopathy, and irritable bowel syndrome (IBS). Rifaximin has multiple indications, including gastrointestinal, orphan, and antibacterial. It belongs to the rifamycin class, acetate class, lactam class, organohexacyclic, macrocyclic, semi-synthetic derivatives, and cyclic ketals. Rifaximin is a semi-synthetic, rifamycin-based, non-systemic antibiotic, meaning it does not cross the gastrointestinal wall into the bloodstream like other oral antibiotics. It has multiple indications for treating traveler's diarrhea caused by Escherichia coli; reducing the risk of relapse of overt hepatic encephalopathy; and treating diarrhea-predominant irritable bowel syndrome (IBS-D) in adult men and women. It is marketed by Salix Pharmaceuticals under the brand name Xifaxan. Rifaximin is a rifamycin-based antibacterial drug. Rifaximin is a poorly absorbed antibiotic used to treat and prevent traveler's diarrhea. At high doses, it can also be used to prevent hepatic encephalopathy in patients with advanced liver disease and to treat diarrhea in patients with irritable bowel syndrome. Rifaximin is poorly absorbed orally and has not been found to cause abnormal liver function or clinically significant liver damage. Rifaximin has been reported in bovine (Bos taurus) with relevant data. Rifaximin is an orally administered, semi-synthetic, non-systemic antibiotic derived from rifamycin SV and possesses antibacterial activity. Rifaximin binds to the β subunit of bacterial DNA-dependent RNA polymerase, inhibiting bacterial RNA synthesis and bacterial cell growth. Due to poor absorption, its antibacterial activity is primarily limited to the gastrointestinal tract. Rifaximin is a synthetic rifamycin derivative and antibacterial agent used to treat gastroenteritis caused by Escherichia coli infection. It can also be used to treat hepatic encephalopathy. See also: Cefazolin sodium (note moved here). Drug Indications Rifaximin is approved by the FDA for multiple indications: treatment of traveler's diarrhea caused by non-invasive strains of E. coli (≥12 years of age); reduction of relapses of overt hepatic encephalopathy in patients ≥18 years of age; and in May 2015, rifaximin was approved for the treatment of diarrhea-predominant irritable bowel syndrome (IBS-D) in adult men and women. FDA Label Mechanism of Action Rifaximin inhibits RNA synthesis in susceptible bacteria by binding to the β subunit of bacterial deoxyribonucleic acid (DNA)-dependent RNA polymerase. This binding blocks RNA translocation, thereby terminating transcription. Pharmacodynamics Rifaximin is a structural analogue of rifampin and is a non-systemic, gastrointestinal targeted antibiotic. Its non-systemic nature stems from the pyridine-imidazole ring introduced into its structure, preventing absorption. Rifaximin works by inhibiting bacterial ribonucleic acid (RNA) synthesis and helps restore gut microbiota balance. Other studies have also shown that rifaximin is a pregnane X receptor (PXR) activator. Since PXR is responsible for inhibiting the pro-inflammatory transcription factor NF-κB, which is suppressed in inflammatory bowel disease (IBD), rifaximin has been shown to be effective in treating diarrhea-predominant irritable bowel syndrome (IBS-D). Mechanism of action: Rifaximin specifically binds to the β subunit (rpoB) of bacterial RNA polymerase, blocking RNA chain synthesis at the transcription initiation stage, inhibiting bacterial gene expression and protein synthesis, and exerting a bactericidal effect [1]. Indications: Used to treat traveler's diarrhea (caused by enterotoxigenic Escherichia coli), Clostridium difficile-associated diarrhea and diarrhea-predominant irritable bowel syndrome (non-infectious) [2]. Resistance mechanism: mainly point mutations in the rpoB gene (commonly found in codons 526 and 528). 531), leading to changes in drug binding sites and reduced affinity [1]
- Advantages of administration: High local concentration in the intestine after oral administration, extremely low systemic absorption, and low incidence of systemic adverse reactions (<2%) [3]
- Use in special populations: Safe for pregnant and lactating women; no dose adjustment is required for patients with hepatic or renal insufficiency [2]

C43H51N3O11

785.88

785.352

C, 65.72; H, 6.54; N, 5.35; O, 22.39

80621-81-4

6436173

Yellow to orange solid powder

1.4±0.1 g/cm3

200-205ºC(dec)

1.634

3.22

5

12

3

57

1590

9

CC1=CC(N2C=C1)=NC3=C2C(NC(/C(C)=C\C=C\[C@H](C)[C@H](O)[C@@H](C)[C@@H](O)[C@@H](C)[C@H](OC(C)=O)[C@@H](C)[C@H](OC)/C=C\O[C@@](C4=O)(C)O5)=O)=C(O)C6=C(O)C(C)=C5C4=C63

NZCRJKRKKOLAOJ-XRCRFVBUSA-N

InChI=1S/C43H51N3O11/c1-19-14-16-46-28(18-19)44-32-29-30-37(50)25(7)40-31(29)41(52)43(9,57-40)55-17-15-27(54-10)22(4)39(56-26(8)47)24(6)36(49)23(5)35(48)20(2)12-11-13-21(3)42(53)45-33(34(32)46)38(30)51/h11-18,20,22-24,27,35-36,39,48-51H,1-10H3,(H,45,53)/b12-11+,17-15+,21-13-/t20-,22+,23+,24+,27-,35-,36+,39+,43-/m0/s1

[(7S,9E,11S,12R,13S,14R,15R,16R,17S,18S,19E,21Z)-2,15,17,36-tetrahydroxy-11-methoxy-3,7,12,14,16,18,22,30-octamethyl-6,23-dioxo-8,37-dioxa-24,27,33-triazahexacyclo[23.10.1.14,7.05,35.026,34.027,32]heptatriaconta-1(35),2,4,9,19,21,25(36),26(34),28,30,32-undecaen-13-yl] acetate

BRN-3584528; L-105; BRN 3584528; L105; BRN3584528; L 105SV; Fatroximin; Normix; Rifacol; Rifamycin L 105; Rifaxidin; Rifaximin; Rifaxin; Ritacol; Rifaximin; trade names: RCIFAX, Rifagut; Xifaxan; Zaxine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 3 mg/mL (3.82 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 3 mg/mL (3.82 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 2.08 mg/mL (2.65 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)