Rifamycin Derivatives Research Articles

Rifamycin antibiotics—new compounds and synthetic methods. Part 4: Study of the reaction of 3-formylrifamycin SV with secondary amines and ketones

In the fourth part of an ongoing study dealing with the search for new rifamycin antibiotics, the reaction of 3-formylrifamycin SV (1) with secondary amines (a.o. pyrrolidine) and ketones of general structure R1–CH2–CO–R2 (R1=H or alkyl and R2=alkyl or aryl) has been investigated. A new synthetic method for the preparation of stable zwitterionic rifamycin derivatives with α,β-unsaturated pyrrolidinium substituents at C3 of the naphthol chromophore has been developed. The structures of the isolated microcrystalline rifamycins and a general mechanism have been proposed on the basis of mass spectrometry results, FTIR, 1D and 2D NMR as well as TLC methods. Rifamycins displaying an aromatic substituent within the pyrrolidinium fragment at C3 displayed a good stability against hydrolysis and proved, in preliminary tests, to be active against TB and MOTT, the activity being for some particular strains (Myc. tbc. Bovis and Myc. scrofulaceum) higher than that observed with the reference drugs (RMP, RBT).

Synthetic Biology in Action: Developing a Drug Against MDR-TB.

The amalgamation of the research efforts of biologists, chemists and geneticists led by scientists at the Department of Zoology, University of Delhi has resulted in the development of a novel rifamycin derivative; 24-desmethylrifampicin, which is highly effective against multi-drug resistant (MDR) strains of Mycobacterium tuberculosis. The production of rifamycin analogue was facilitated by genetic-synthetic strategies that have opened an interdisciplinary route for the development of more such rifamycin analogues aiming at a better therapeutic potential. The results of this painstaking effort of nearly 25years of a team of students and scientists led by Professor Rup Lal have been recently published in the Journal of Biological Chemistry (www.jbc.org/content/289/30/21142.long). This strategy can now find applications for developing newer rifamycin analogues that can be harnessed to overcome the problem of MDR, extensively drug resistant (XDR) and totally drug resistant (TDR) M. tuberculosis.

Structural basis of Rifampin resistance in bacterial RNA polymerase (939.1)

For over four decades, Rifampin (Rif), a semi‐synthetic derivative of Rifamycin, has been used as a first line antibiotic treatment of tuberculosis and is the cornerstone of current short‐term tuberculosis treatment. The mode of action involves tight Rif binding to a beta subunit of bacterial RNA polymerase (RNAP) to inhibit RNA transcription. Although many Rif resistant (RifR) strains with mutations in the Rif‐binding pocket can be isolated in bacterial culture, only three specific RifR mutations account for over 80 % of Mycobacterium tuberculosis (MTB) RifR strains in clinical isolates due to RifR associated fitness costs.In this study, we have determined the X‐ray crystal structures of the E. coli RNAP RifR mutants each having one of three major RifR mutations found in clinical isolates. Each RifR RNAP structure shows a unique conformation of the Rif binding pocket and their structural deviations from the wild‐type Rif binding pocket are consistent with their Rif resistances suggesting that the RifR results from alternating the shape complementary between the Rif binding pocket and Rif rather than disrupting their hydrophilic and hydrophobic interactions. This study provides an important step for understanding the structure−activity relationship of Rif against RifR RNAP inhibition and toward developing superior Rif analogues against RifR MTB.Grant Funding Source: Supported by NIH

Antibacterial activity of rifamycins for M. smegmatis with comparison of oxidation and binding to tear lipocalin

A mutant of Mycobacterium smegmatis is a potential class I model substitute for Mycobacterium tuberculosis. Because not all of the rifamycins have been tested in this organism, we determined bactericidal profiles for the 6 major rifamycin derivatives. The profiles closely mirrored those established for M. tuberculosis. Rifalazil was confirmed to be the most potent rifamycin. Because the tuberculous granuloma presents a harshly oxidizing environment we explored the effects of oxidation on rifamycins. Mass spectrometry confirmed that three of the six major rifamycins showed autoxidation in the presence of trace metals. Oxidation could be monitored by distinctive changes including isosbestic points in the ultraviolet–visible spectrum. Oxidation of rifamycins abrogated anti-mycobacterial activity in M. smegmatis. Protection from autoxidation was conferred by binding susceptible rifamycins to tear lipocalin, a promiscuous lipophilic protein. Rifalazil was not susceptible to autoxidation but was insoluble in aqueous solution. Solubility was enhanced when complexed to tear lipocalin and was accompanied by a spectral red shift. The positive solvatochromism was consistent with robust molecular interaction and binding. Other rifamycins also formed a complex with lipocalin, albeit to a lesser extent. Protection from oxidation and enhancement of solubility with protein binding may have implications for delivery of select rifamycin derivatives.

In vitro activity of rifaximin against isolates from patients with small intestinal bacterial overgrowth

Rifaximin, a non-absorbable rifamycin derivative, has published clinical efficacy in the alleviation of symptoms in patients with irritable bowel syndrome (IBS). Small intestinal bacterial overgrowth (SIBO) is associated with the pathogenesis of IBS. This study describes for the first time the antimicrobial effect of rifaximin against SIBO micro-organisms from humans. Fluid was aspirated from the third part of the duodenum from 567 consecutive patients; quantitative cultures diagnosed SIBO in 117 patients (20.6%). A total of 170 aerobic micro-organisms were isolated and the in vitro efficacy of rifaximin was studied by (i) minimum inhibitory concentration (MIC) testing by a microdilution technique and (ii) time–kill assays using bile to simulate the small intestinal environment. At a breakpoint of 32μg/mL, rifaximin inhibited in vitro 85.4% of Escherichia coli, 43.6% of Klebsiella spp., 34.8% of Enterobacter spp., 54.5% of other Enterobacteriaceae spp., 82.6% of non-Enterobacteriaceae Gram-negative spp., 100% of Enterococcus faecalis, 100% of Enterococcus faecium and 100% of Staphylococcus aureus. For the time–kill assays, 11 E. coli, 15 non-E. coli Gram-negative enterobacteria and three E. faecalis isolates were studied. Rifaximin produced a >3log10 decrease in the starting inoculum against most of the tested isolates at 500μg/mL after 24h of growth. The results indicate that rifaximin has a potent effect on specific small bowel flora associated with SIBO. This conclusion should be regarded in light of the considerable time–kill effect at concentrations lower than those achieved in the bowel lumen after administration of conventional doses in humans.

Rifaximin-Mediated Changes to the Epithelial Cell Proteome: 2-D Gel Analysis

Rifaximin is a semi-synthetic rifamycin derivative that is used to treat different conditions including bacterial diarrhea and hepatic encephalopathy. Rifaximin is of particular interest because it is poorly adsorbed in the intestines and has minimal effect on colonic microflora. We previously demonstrated that rifaximin affected epithelial cell physiology by altering infectivity by enteric pathogens and baseline inflammation suggesting that rifaximin conferred cytoprotection against colonization and infection. Effects of rifaximin on epithelial cells were further examined by comparing the protein expression profile of cells pretreated with rifaximin, rifampin (control antibiotic), or media (untreated). Two-dimensional (2-D) gel electrophoresis identified 36 protein spots that were up- or down-regulated by over 1.7-fold in rifaximin treated cells compared to controls. 15 of these spots were down-regulated, including annexin A5, intestinal-type alkaline phosphatase, histone H4, and histone-binding protein RbbP4. 21 spots were up-regulated, including heat shock protein (HSP) 90α and fascin. Many of the identified proteins are associated with cell structure and cytoskeleton, transcription and translation, and cellular metabolism. These data suggested that in addition to its antimicrobial properties, rifaximin may alter host cell physiology that provides cytoprotective effects against bacterial pathogens.

Concentration of clarithromycin and 14-R-hydroxy-clarithromycin in plasma of patients with Mycobacterium avium complex infection, before and after the addition of rifampicin.

Clarithromycin (CAM) and rifampicin (RFP) have both been recognized to be effective antibiotic agents against Mycobacterium avium complex (MAC) infection. Rifamycin derivatives including RFP and rifabutin modulate the CAM metabolism by inducing the hepatic cytochrome p-450 3A4. To clarify the effect of RFP on the CAM metabolism, we measured the plasma concentration of CAM and 14-R-hydroxyclarithromycin (M-5), the major metabolite of CAM, in 9 patients suffering from MAC infection before and after the addition of RFP. After the addition of RFP, the mean plasma concentration of CAM significantly decreased, while that of M-5 did not. In addition, the amount of CAM + M-5 concentration also significantly decreased. As M-5 is less effective against MAC infection than CAM, more attention should thus be paid to the plasma CAM concentration in patients administered CAM and RFP concomitantly.

Bactericidal Activity Does Not Predict Sterilizing Activity: The Case of Rifapentine in the Murine Model of Mycobacterium ulcerans Disease

BackgroundSince 2004, treatment of Mycobacterium ulcerans disease, or Buruli ulcer, has shifted from surgery to daily treatment with streptomycin (STR) + rifampin (RIF) for 8 weeks. For shortening treatment duration, we tested the potential of daily rifapentine (RPT), a long-acting rifamycin derivative, as a substitute for RIF.Methodology/Principal FindingsBALB/c mice were infected with M. ulcerans in the right hind footpad and treated either daily (7/7) with STR+RIF or five days/week (5/7) with STR+RIF or STR+RPT for 4 weeks, beginning 28 days after infection when CFU counts were 4.88±0.51.The relative efficacy of the drug treatments was compared by footpad CFU counts during treatment and median time to footpad swelling after treatment cessation as measure of sterilizing activity. All drug treatments were bactericidal. After 1 week of treatment, the decline in CFU counts was significantly greater in treated mice but not different between the three treated groups. After 2 weeks of treatment, the decline in CFU was greater in mice treated with STR+RPT 5/7 than in mice treated with STR+RIF 7/7 and STR+RIF 5/7. After 3 and 4 weeks of treatment, CFU counts were nil in mice treated with STR+RPT and reduced by more than 3 and 4 logs in mice treated with STR+RIF 5/7 and STR+RIF 7/7, respectively. In sharp contrast to the bactericidal activity, the sterilizing activity was not different between all drug regimens although it was in proportion to the treatment duration.Conclusions/SignificanceThe better bactericidal activity of daily STR+RIF and especially of STR+RPT did not translate into better prevention of relapse, possibly because relapse-freecure after treatment of Buruli ulcer is more related to the reversal of mycolactone-induced local immunodeficiency by drug treatment rather than to the bactericidal potency of drugs.

India’s Latent Foe: Multi Drug Resistant Tuberculosis

Tuberculosis (TB), an infectious bacterial disease, is caused by Mycobacterium tuberculosis. An effective drug for the control of tuberculosis is rifamycin which is clinically used in combination with ethambutol, isoniazid and pyrazinamide. Generally, a six month treatment regime is competent enough to cure tuberculosis, but due to inappropriate treatment and a non committal approach of the patients towards the treatment, there has been a drastic rise in the cases of Multi drug resistant (MDR) mycobacterial strains. Such cases, have increased in drastic numbers all across the globe especially in the developing and under developed countries, where severe lack of awareness has fuelled this ominous a phenomena. Besides this, the incidence of TB and HIV infection seem to go hand in hand, as a suppressed immune system makes the patient more amenable to other bacterial infections. India alone harbours around a million patients coinfected with HIV and M. tuberculosis. While several efforts are being made to tackle this problem, nothing of great significance has been brought out. Rifamycin, the classically used drug for curing TB is produced by an actinobacterium Amycolatopsis mediterranei. However, rifamycin and its semisynthetic derivatives are largely becoming ineffective against the drug resistant forms of mycobacteria. Eversince the commercial production of rifamycin in 1962, it has been a boon to the mankind in its ability to control TB. With immense efforts of the researchers, the yield of this wonder drug from A. mediterranei was increased from a meagre 500 mg/L in 1962 to up to 24 g/L through classical strain improvement methods. However, with the incidence of 73,000 cases of MDR-TB in India, and around a million across the world, there is an urgent need to develop rifamycin analogs or completely new drugs effective against MDR-TB strains. So far only six semisynthetic derivatives of rifamycin B could be produced by modifying the naphthoquinone ring at positions C3 and C4, beyond which, modifications are not possible. Alternative to this, could be the combinatorial approach of deleting, swapping or inactivating modules or domains of rifamycin biosynthetic gene cluster (also called polyketide synthase gene cluster). This approach has been successful with erythromycin polyketide gene cluster, in Saccharospora erythraea, but has not been applied to rifamycin polyketide synthase gene cluster, yet. Thus, in future, emphasis should be laid on this approach to tackle the problem of MDR-TB.

Rifamycin antibiotics—new compounds and synthetic methods. Part 3: Study of the reaction of 3-formylrifamycin SV with primary amines and ketones

In the third stage of our study concerning the search for new antibacterial rifamycin antibiotics, the reactions of 3-formylrifamycin SV (1) with a range of primary alkylamines and ketones of general structure R1–CH2–CO–R2 (R1H or alkyl and R2alkyl or aryl) has been investigated. A new synthetic method for the preparation of a new group of rifamycin derivatives with an α,β-unsaturated imine substituent at C-3 has been developed.These compounds showed a tendency to reversibly isomerise in organic solvents and, in the presence of water, to rapidly hydrolyse. The structures of four isolated microcrystalline compounds 2, 3, 4, 5 and a reaction's mechanism have been proposed on the basis of mass spectrometry results as well as (1D) and (2D) 1H and 13C NMR analysis. The new synthetic route reported herein is a promising pathway to new reactive rifamycins displaying broader capabilities than the plain 3-formylrifamycin SV.

Characterization of rifamycin‐resistant Mycobacterium tuberculosis RNA polymerases

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB) where over 9 million people are infected globally and approximately 2 million lives are claimed annually. Rifampin (a semi‐synthetic rifamycin derivative) inhibits the MTB RNA polymerase (RNAP) and is one of the first‐line anti‐TB drugs. However rifamycin‐resistant (RifR) mutations are frequent and the individual substitutions of three β‐subunit residues of the RNAP (Asp435, His445, and Ser450) together account for 84% of MTB RifR strains found in clinical isolates. To better understand the interaction between rifamycins and wild‐type/RifR mutant MTB RNAPs, the IC50 values of known/novel rifamycins against RifR MTB RNAPs were determined. The inhibition of the different MTB RNAPs by rifamycins was investigated via dose‐response studies with an in vitro rolling circle transcription assay. Rifamycins bind tightly to the wild‐type MTB RNAP and inhibit the enzyme in the 10−9 M (nM) range. Whereas for the RifR mutants, a dramatic ~102–105 fold loss of affinity for rifamycins was observed where these different mutants were inhibited at much higher concentrations of rifamycins (10s–100s μM range). A direct binding assay is being developed to determine the equilibrium binding constant (Kd) values to further characterize the rifamycin·RNAP interactions.

High-resolution melting analysis of the single nucleotide polymorphism hot-spot region in the rpoB gene as an indicator of reduced susceptibility to rifaximin in Clostridium difficile

Clostridium difficile, a Gram-positive, spore-forming, anaerobic bacterium, is the main causative agent of hospital-acquired diarrhoea worldwide. In addition to metronidazole and vancomycin, rifaximin, a rifamycin derivative, is a promising antibiotic for the treatment of recurring C. difficile infections (CDI). However, exposure of C. difficile to this antibiotic has led to the development of rifaximin-resistance due to point mutations in the β-subunit of the RNA polymerase (rpoB) gene. In the present study, 348 C. difficile strains with known PCR-ribotypes were investigated for respective single nucleotide polymorphisms (SNPs) within the proposed rpoB hot-spot region by using high-resolution melting (HRM) analysis. This method allows the detection of SNPs by comparing the altered melting behaviour of dsDNA with that of wild-type DNA. Discrimination between wild-type and mutant strains was enhanced by creating heteroduplexes by mixing sample DNA with wild-type DNA, leading to characteristic melting curve shapes from samples containing SNPs in the respective rpoB section. In the present study, we were able to identify 16 different rpoB sequence-types (ST) by sequencing analysis of a 325 bp fragment. The 16 PCR STs displayed a total of 24 different SNPs. Fifteen of these 24 SNPs were located within the proposed 151 bp SNP hot-spot region, resulting in 11 different HRM curve profiles (CP). Eleven SNPs (seven of which were within the proposed hot-spot region) led to amino acid substitutions associated with reduced susceptibility to rifaximin and 13 SNPs (eight of which were within the hot-spot region) were synonymous. This investigation clearly demonstrates that HRM analysis of the proposed SNP hot-spot region in the rpoB gene of C. difficile is a fast and cost-effective method for the identification of C. difficile samples with reduced susceptibility to rifaximin and even allows simultaneous SNP subtyping of the respective C. difficile isolates.

Whole Genome Sequence of the Rifamycin B-Producing Strain Amycolatopsis mediterranei S699

Amycolatopsis mediterranei S699 is an actinomycete that produces an important antibiotic, rifamycin B. Semisynthetic derivatives of rifamycin B are used for the treatment of tuberculosis, leprosy, and AIDS-related mycobacterial infections. Here, we report the complete genome sequence (10.2 Mb) of A. mediterranei S699, with 9,575 predicted coding sequences.

Rifaximin for the treatment of acute infectious diarrhea.

Rifaximin is a nonabsorbable rifamycin derivative with an excellent safety profile and a broad spectrum of antimicrobial activity against a variety of enteropathogens causing acute infectious diarrhea. After oral ingestion, its bioavailability is known to be less than 0.4%, and it has a low potential for significant drug interactions. In the treatment of travelers' diarrhea caused by noninvasive diarrheagenic Escherichia coli, it has been demonstrated that rifaximin significantly shortens the duration of diarrhea and has an efficacy similar to that of ciprofloxacin. Moreover, according to two randomized placebo-controlled trials, prophylactic treatment with rifaximin reduced the risk of developing travelers' diarrhea by more than 50% compared with the placebo group. For the treatment of acute diarrhea unrelated to travel, a short course of rifaximin significantly reduced the duration of diarrhea, and its overall efficacy was comparable to that of ciprofloxacin. The discrepancy between the invitro and invivoantimicrobial activities of rifaximin, however, and the clinical implication of the rapid appearance of bacterial resistance, must be further elucidated. In conclusion, this gut-selective antibiotic seems to be a promising option for the treatment of acute infectious diarrhea secondary to noninvasive E. coli and also appears to be effective in chemoprophylaxis for travelers' diarrhea.

Mechanism of bacterial regrowth at the sites of infection in Mycobacterium avium complex-infected mice during treatment with chemotherapeutic agents

Although various antimicrobial drugs show appreciable bactericidal activity in the early phase (2 to 4 weeks after infection) of Mycobacterium avium complex (MAC) infections in mice, no drug, as far as we know, can continue to exert the growth inhibiting activity against the bacteria at the site of infection in the progressed stage. Here, we studied the mechanisms of the bacterial regrowth which usually starts around 2-4 weeks after infection. First, the changes in the level of TNF-alpha, IFN-gamma, IL-6 and IL-10 in the lungs and spleen during the course of MAC infections was examined. Tissue levels of TNF-alpha and IL-10 increased around weeks 2 to 4, then rapidly decreased thereafter, and returned to the normal levels by week 8, while levels of IFN-gamma and IL-6 remained very low throughout the observation period. In this experiment, the bacterial CFUs rapidly decreased during the first 2 weeks of the treatment with a rifamycin derivative, KRM-1648, and thereafter the regrowth of the organisms was observed even in mice treated continuously with KRM-1648, although the rate of bacterial growth in the treated mice was much lower than in untreated control mice. Second, effect of either anti-TGF-beta or anti-IL-10 antibody on intracellular growth of MAC in human monocytes cultured in vitro in the medium with or without addition of TNF-alpha or IFN-gamma were examined. Anti-TGF-beta and anti-IL-10 antibodies potently reduced the bacterial growth in monocytes. Effects of TNF-alpha and IFN-gamma in reducing the bacterial growth was potentiated by the addition of either anti-TGF-beta or anti-IL-10 antibody. Third, anti-IL-10 antibody augmented to some extent the chemotherapeutic efficacy of KRM-1648 against MAC infection, when the drug was given to mice at weeks 2 and 4 after infection. From these results, it is suggested that IL-10 derived from MAC-infected macrophages in response to stimulation with some bacterial components, such as lipoarabinomannan, might downregulate the antimicrobial function of host macrophages against MAC.

Establishment of the quantitative intratracheal infection model of experimental murine mycobacteriosis

An intratracheal infection method (IT) of experimental murine mycobacteriosis was developed for an in vivo study of antimycobacterial agents. IT-model with either of M. bovis, M. tuberculosis or M. intracellulare exhibited a much more distinct lung-specific infection than intravenous model (i.v.) with the same dose of respective mycobacterial strains. Through a series of comparative studies of benzoxazinorifamycin (KRM) with rifabutin (RBT) or rifampicin (RFP) against murine tuberculosis models, therapeutic efficacy in the lungs of IT-model was superior to those of i.v.-model with the same dose of respective drugs (Fig. 2). In IT-model of M. bovis Ravenel infection, three rifamycin derivatives gave "distinctive dose-response curves" in the correlation of dose sizes with the mean survival times or "log10 CFU/lungs reductions". Moreover, based on the results of "log10 CFU reductions" in different organs in M. tuberculosis Kurono infection models, "characteristic in vivo activity patterns of each rifamycin" was obtained. An outline of the new methods for evaluating and characterizing the in vivo activities of antimycobacterial agents was presented in Fig. 3. This IT-model may be useful not only for the in vivo assessment of antimycobacterial agents but also for the comparison of virulence among various mycobacterial strains.

Antibiotic Therapy in Inflammatory Bowel Disease: A Systematic Review and Meta-Analysis

The etiology of inflammatory bowel disease (IBD) is unknown but may relate to an unidentified bacterial pathogen or an immunological reaction to gut microbiota. Antibiotics have therefore been proposed as a therapy for Crohn's disease (CD) and ulcerative colitis (UC) to induce remission in active disease to prevent relapse. Current data are conflicting and we therefore conducted a systematic review of randomized controlled trials (RCTs) evaluating antibiotics in IBD. Only parallel group RCTs were considered eligible. Studies with adult patients receiving any dose of therapy for at least 7 days and up to 16 weeks for active disease, or at least 6 months of follow-up for preventing relapse in quiescent disease were analyzed. We included any antibiotics alone or in combination using predefined definitions of remission and relapse. Two reviewers independently assessed eligibility and extracted data. The primary outcome was remission or relapse using an intention-to-treat methodology. The data were summarized using relative risk (RR) and pooled using a random effects model. For active CD, there were 10 RCTs involving 1,160 patients. There was a statistically significant effect of antibiotics being superior to placebo (RR of active CD not in remission=0.85; 95% confidence interval (CI)=0.73-0.99, P=0.03). There was moderate heterogeneity between results (I(2)=48%) and a diverse number of antibiotics were tested (anti-tuberculosis therapy, macrolides, fluroquinolones, 5-nitroimidazoles, and rifaximin) either alone or in combination. Rifamycin derivatives either alone or in combination with other antibiotics appeared to have a significant effect at inducing remission in active CD. In perianal CD fistula there were three trials evaluating 123 patients using either ciprofloxacin or metronidazole. There was a statistically significant effect in reducing fistula drainage (RR=0.8; 95% CI=0.66-0.98) with no heterogeneity (I(2)=0%) and an number needed to treat 5 (95% CI=3-20). For quiescent CD, there were 3 RCTs involving 186 patients treated with different antibiotics combinations (all including antimycobacterials) vs. placebo. There was a statistically significant effect in favor of antibiotics vs. placebo (RR of relapse=0.62; 95% CI=0.46-0.84), with no heterogeneity (I(2)=0%). In active UC, there were 9 RCTs with 662 patients and there was a statistically significant benefit for antibiotics inducing remission (RR of UC not in remission=0.64; 95% CI=0.43-0.96). There was moderate heterogeneity (I(2)=69%) and antibiotics used were all different single or combination drugs. Antibiotic therapy may induce remission in active CD and UC, although the diverse number of antibiotics tested means the data are difficult to interpret. This systematic review is a mandate for further trials of antibiotic therapy in IBD.

Efficacy of Rifaximin Compared with Ciprofloxacin for the Treatment of Acute Infectious Diarrhea: A Randomized Controlled Multicenter Study

Ciprofloxacin has been widely prescribed for acute infectious diarrhea. However, the resistance to this drug is increasing. Rifaximin is a novel but poorly absorbed rifamycin derivative. This study evaluated and compared the efficacies of rifaximin and ciprofloxacin for the treatment of acute infectious diarrhea. We performed a randomized controlled multicenter study in Korea. Patients with acute diarrhea were enrolled and randomized to receive rifaximin or ciprofloxacin for 3 days. The primary efficacy endpoint was the time to last unformed stool (TLUS). Secondary endpoints were enteric wellness (reduction of at least 50% in the number of unformed stools during 24-hour postenrollment intervals), general wellness (subjective feeling of improvement), and proportion of patients with treatment failure. Intent-to-treat analysis (n=143) showed no significant difference between the rifaximin and ciprofloxacin groups in the mean TLUS (36.1 hours vs 43.6 hours, p=0.163), enteric wellness (49% vs 57%, p=0.428), general wellness (67% vs 78%, p=0.189), or treatment failure rate (9% vs 12%, p=0.841). The adverse events did not differ significantly between the two groups. These results suggest that rifaximin is as safe and effective as ciprofloxacin in the treatment of acute infectious diarrhea.

Rifaximin modulates the colonic microbiota of patients with Crohn's disease: an in vitro approach using a continuous culture colonic model system

Rifaximin, a rifamycin derivative, has been reported to induce clinical remission of active Crohn's disease (CD), a chronic inflammatory bowel disorder. In order to understand how rifaximin affects the colonic microbiota and its metabolism, an in vitro human colonic model system was used in this study. We investigated the impact of the administration of 1800 mg/day of rifaximin on the faecal microbiota of four patients affected by colonic active CD [Crohn's disease activity index (CDAI > 200)] using a continuous culture colonic model system. We studied the effect of rifaximin on the human gut microbiota using fluorescence in situ hybridization, quantitative PCR and PCR-denaturing gradient gel electrophoresis. Furthermore, we investigated the effect of the antibiotic on microbial metabolic profiles, using (1)H-NMR and solid phase microextraction coupled with gas chromatography/mass spectrometry, and its potential genotoxicity and cytotoxicity, using Comet and growth curve assays. Rifaximin did not affect the overall composition of the gut microbiota, whereas it caused an increase in concentration of Bifidobacterium, Atopobium and Faecalibacterium prausnitzii. A shift in microbial metabolism was observed, as shown by increases in short-chain fatty acids, propanol, decanol, nonanone and aromatic organic compounds, and decreases in ethanol, methanol and glutamate. No genotoxicity or cytotoxicity was attributed to rifaximin, and conversely rifaximin was shown to have a chemopreventive role by protecting against hydrogen peroxide-induced DNA damage. We demonstrated that rifaximin, while not altering the overall structure of the human colonic microbiota, increased bifidobacteria and led to variation of metabolic profiles associated with potential beneficial effects on the host.

A bioequivalence comparison of two formulations of rifampicin (300- vs 150-mg capsules): An open-label, randomized, two-treatment, two-way crossover study in healthy volunteers

Background: Rifampicin is a semisynthetic antibiotic derivative of rifamycin used worldwide for the treatment of various forms of tuberculosis. Objective: The objective of this study was to compare, under fasting conditions in healthy volunteers, the rate and extent of absorption of a generic rifampicin capsule in oral dosage form versus the proprietary equivalent formulation for the purpose of registration approval of the test formulation. Methods: This was an open-label, randomized, 2-treatment, 2-way crossover study with an 8-week washout period between the 2 study arms. Healthy volunteers received a 300-mg capsule of the test formulation (Idaman Pharma Manufacturing Sdn. Bhd.) or two 150-mg capsules of the reference formulation. Blood samples were collected predose and at 45 minutes and 1.25, 1.5, 2, 2.25, 2.5, 3, 3.5, 4, 6, 8, 10, 12, and 24 hours postdose. Plasma concentrations of rifampicin and its metabolite, 25-desacetyl rifampicin, were analyzed using a validated HPLC method. The formulations were considered bioequivalent if the 90% CIs for C max and AUC were within the predetermined bioequivalence range (80%–125%, according to the guidelines of the US Food and Drug Administration, or 75%–133% for Cmax only, as set by the European Commission-European Medicines Agency and the National Pharmaceutical Control Bureau of Malaysia). Tolerability was assessed by verbally questioning subjects regarding their well-being and any feelings of discomfort. All events reported by the subjects (serious or mild) were recorded on adverse-event forms. Results: Fourteen healthy subjects (10 males, 4 females) with a mean age of 22.6 years (range, 20–28 years) and a mean body mass index of 22.2 kg/m 2 (range, 18.3-29.9 kg/m2) were enrolled in the study; all 14 completed the trial as outlined in the protocol. The mean values for C max, T max , AUC 0–24, and AUC 0−∞) with the test formulation of rifampicin were 7.20 μg/mL, 1.32 hours, 37.12 μg/mL · h, and 39.69 μg/mL · h, respectively; for the reference formulation, the values were 7.65 μg/mL, 1.71 hours, 38.92 μg/mL · h, and 42.24 μg/ mL · h. For 25-desacetyl rifampicin, the mean values for C max, T max, AUC 0−24, and AUC 0−∞) with the test formulation were 0.63 μg/mL, 3.45 hours, 4.92 μg/mL · h, and 6.27 μg/mL · h; for the reference formulation, the values were 0.7 μg/mL, 3.27 hours, 5.23 μg/mL · h, and 6.84 μg/mL · h. For rifampicin, the 90% CIs for the test formulation/reference formulation ratio for the logarithmic transformations of both C max and AUC 0−∞) were within the bioequivalence limit of 80% to 125% (80.9109.7 and 80.7–103.2, respectively). For 25-desacetyl rifampicin, the 90% CI for the test formulation/reference formulation ratio for the logarithmic transformations of AUC 0–24 (80.0–104.7) was within the bioequivalence limit of 80% to 125%. However, the 90% CI for C max (78.4–102.2) was outside this limit but still within the acceptance limit for Cmax when adhering to the bioequivalence range of 75% to 133%. No adverse events were reported during the study. Conclusions: This study found that the 300-mg test capsule and the 150-mg reference capsules of rifampicin met the regulatory criteria for assuming bioequivalence in these fasting healthy volunteers. Both formulations appeared to be well tolerated in the population studied.