Pharmacokinetic study of rifampicin in the body of pregnant animals]

The study on distribution of 14C-rifampicin administered intramuscularly to pregnent animals showed that its concentrations in the blood, liver, kidneys, lungs and other organs did not practically change from those in nonpregnant animals. The concentration of 14C-rifampicin in the fetus organs was much lower than that in the organs of the adult animals. The liver and kidneys of the pregnant animals, as well as the fetus though to a less extent had a capacity for metabolism of 14C-rifampicin. The following products of biotransformation were detected: N-oxide of rifampicin, 25-deacetylrifampicin, 3-formylrifamycin SV and rifamycin SV.     

Isolation and characterization of 27-O-demethylrifamycin SV methyltransferase provides new insights into the post-PKS modification steps during the biosynthesis of the antitubercular drug rifamycin B by Amycolatopsis mediterranei S699

The gene rif orf14 in the rifamycin biosynthetic gene cluster of Amycolatopsis mediterranei S699, producer of the antitubercular drug rifamycin B, encodes a protein of 272 amino acids identified as an AdoMet: 27-O-demethylrifamycin SV methyltransferase. Frameshift inactivation of rif orf14 generated a mutant of A. mediterranei S699 that produces no rifamycin B, but accumulates 27-O-demethylrifamycin SV (DMRSV) as the major new metabolite, together with a small quantity of 27-O-demethyl-25-O-desacetylrifamycin SV (DMDARSV). Heterologous expression of rif orf14 in Escherichia coli yielded a 33.8-kDa polyhistidine-tagged polypeptide, which efficiently catalyzes the methylation of DMRSV to rifamycin SV, but not that of DMDARSV or rifamycin W. 27-O-Demethylrifamycin S was methylated poorly, if at all, by the enzyme to produce rifamycin S. The purified enzyme does not require a divalent cation for catalytic activity. While Ca(2+) or Mg(2+) inhibits the enzyme activity slightly, Zn(2+), Ni(2+), and Co(2+) are strongly inhibitory. The K(m) values for DMRSV and S-adenosyl-L-methionine (AdoMet) are 18.0 and 19.3 microM, respectively, and the K(cat) is 87s(-1). The results indicate that DMRSV is a direct precursor of rifamycin SV and that acetylation of the C-25 hydroxyl group must precede the methylation reaction. They also suggest that rifamycin S is not the precursor of rifamycin SV in rifamycin B biosynthesis, but rather an oxidative shunt-product.     

Effects of inhibitors on growth and ribosomal-RNA synthesis in cultured spinach leaf discs

In single isotope labelling experiments it was found that rifamycin SV (100 μg/ml) but not rifampicin (100 μg/ml) inhibited cytoplasmic ribosomal-RNA synthesis. Dual-isotope labelling experiments established that rifamycin SV inhibited light-stimulated chloroplast ribosomal-RNA synthesis to the same extent. Light-stimulated chloroplast ribosomal-RNA synthesis was specifically inhibited by streptolydigin(50 μg/ml), lincomycin(100μg/ml) and chloramphenicol(10μg/ml).     

Oxygen Enhancement of bactericidal activity of rifamycin SV on Escherichia coli and aerobic oxidation of rifamycin SV to rifamycin S catalyzed by manganous ions: the role of superoxide

Oxygen enhanced the bactericidal activity of rifamycin SV to Escherichia coli K12. Anaerobically grown cells, which had a low level of superoxide dismutase, were more susceptible to the bactericidal activity than aerobically grown cells, which contained a high level of superoxide dismutase. Oxygen also enhanced the inhibition of RNA polymerase activity of rifamycin SV, when Mn2+ was used as a cofactor. Rifamycin S was reduced to rifamycin SV by NADPH catalyzed by cell-free extracts of Escherichia coli K12. These results indicate that the inhibition of bacterial growth by rifamycin SV is due to the production of active species of oxygen resulting from the oxidation-reduction cycle of rifamycin SV in the cells. The aerobic oxidation of rifamycin SV to rifamycin S was induced by metal ions, such as Mn2+, Cu2+, and Co2+. The most effective metal ion was Mn2+. In the presence of Mn2+, accompanying the consumption of 1 mol of oxygen and the oxidation of 1 mol of rifamycin SV, 1 mol of hydrogen peroxide and 1 mol of rifamycin S were formed. Superoxide was generated during the autoxidation of rifamycin SV. Superoxide dismutase inhibited the formation of rifamycin S, but scavengers for hydrogen peroxide and the hydroxyl radical did not affect the oxidation. A mechanism of Mn2+-catalyzed oxidation of rifamycin SV is proposed and its relation to bactericidal activity is discussed.     

Rifamycin derivatives: specific inhibitors of nucleic acid polymerases

Rifampicin and three rifamycin SV derivatives with different lipophilic side chains were tested as inhibitors of a number of purified enzymes including the α and αβ forms of RNA-directed DNA polymerase of avian myeloblastosis virus (AMV). $\text{AF}\text{ABDMP}$ (2,5-dimethyl-4-N-benzyl demethyl rifampicin), $\text{AF}\text{013}$ (O-n-octyloxime of 3-formyl rifamycin SV) and C-27 (rifamycin SV with a dicyclohexylalkyl substituted piperidyl ring at the 3-position) at concentrations less than 20 to 40 μg/ml completely inhibited the RNA- and DNA-directed DNA polymerase and RNase H activities of both AMV enzymes. Rifampicin was inactive at 100 μg/ml. When used against a variety of non-polymerizing enzymes such as alkaline phosphatase, glutamate-oxaloacetate transaminase, DNase I, and RNase A, these derivatives were inactive at drug concentrations between 100 and 200 μg/ml. Polynucleotide phosphorylase was inhibited slightly by all three derivatives. These results support the idea that rifamycin SV derivatives with appropriate 3-substituted side-chains are specific inhibitors of nucleic acid polymerizing enzymes.     

Mechanism of action of rifamazine, a member of a new class of (dimeric) rifamycins.

1. Rifamazine (AF/RP) a dimeric rifamycin, is active against bacterial DNA-dependent RNA polymerase and against viral RNA-dependent DNA polymerase. 2. Rifamazine is active also against DNA-dependent RNA polymerase extracted from rifampicin-resistant mutants of Escherichia coli. It does not interfere with enzyme-template interaction or with RNA elongation. It blocks initiation. 3. A comparison is made between the mechanism of action of rifamazine and that of rifampicin, and of AF/013 (octyloxime of 3-formylrifamycin SV), a C-class rifamycin. Our results show that the mechanism of action of rifamazine is more similar to that of rifampicin than to that of the octyloxime derivative. 4. Activity of rifamazine against RNA polymerase from rifampicin-resistant mutants is thought to be due to binding of the dimer to both the rifamycin-specific binding site and to a second weak site.     

Stimulation of microsomal production of reactive oxygen intermediates by rifamycin SV: effect of ferric complexes and comparisons between NADPH and NADH.

Rifamycins are antibacterial antibiotics which are especially useful for the treatment of tuberculosis. Reactive oxygen intermediates are produced in the presence of rifamycin SV and metals such as copper or manganese. Experiments were carried out to evaluate the interaction of rifamycin SV with rat liver microsomes to catalyze the production of reactive oxygen species. At a concentration of 1 mM, rifamycin SV increased microsomal production of superoxide with NADPH as cofactor 3-fold, and with NADH as reductant by more than 5-fold. Rifamycin SV increased rates of H2O2 production by the microsomes twofold with NADPH, and 4- to 8-fold with NADH. In the presence of various iron complexes, microsomes generated hydroxyl radical-like (.OH) species. Rifamycin SV had no effect on NADPH-dependent microsomal .OH production, irrespective of the iron chelate. A striking stimulation of .OH production was found with NADH as the reductant, ranging from 2- to 4-fold with catalyst such as ferric-EDTA and ferric-DTPA to more than 10-fold with ferric-ATP, -citrate, or -histidine. Catalase and competitive .OH scavengers lowered rates of .OH production (chemical scavenger oxidation) and prevented the stimulation by rifamycin. Superoxide dismutase had no effect on the NADH-dependent rifamycin stimulation of .OH production with ferric-EDTA or -DTPA, but was inhibitory with the other ferric complexes. In contrast to the stimulatory effects on production of O2-., H2O2, and .OH, rifamycin SV was a potent inhibitor of microsomal lipid peroxidation. These results show that rifamycin SV stimulates microsomal production of reactive oxygen intermediates, and in contrast to results with other redox cycling agents, is especially effective with NADH as the microsomal reductant. These interactions may contribute to the hepatotoxicity associated with use of rifamycin, and, since alcohol metabolism increases NADH availability, play a role in the elevated toxic actions of rifamycin plus alcohol.     

Resolution of binary mixtures of rifamycin SV and rifampicin by UV/VIS spectroscopy and partial least-squares method (PLS)

A procedure is proposed for the joint determination of rifamycin SV and rifampicin by UV/VIS spectroscopy. A partial least-squares regression (PLS) was used for the resolution of the overlapping spectrophotometric signals from mixtures of the two drugs. The application of a genetic algorithm to select some of the predictor variables allows one to considerably reduce the number of experimental variables, as well as to improve the prediction capacity of the PLS model constructed with these selected potentials. Finally, the methods were applied to the determination of these drugs in biological samples.     

Comparative study of the effect of different rifamycins on bacterial cell metabolism and on the RNA-polymerase reaction in a cell-free system]

The results of the study on the inhibitory effect of a number of rifamycin derivatives, such as rifamycin B, rifamycin O, rifamycin, rifamycin A, 25-desacetylrifampicin and rifampicin are presented. It was shown that rifampicin had the highest inhibitory effect on the synthesis of RNA in the cells of E. coli and Staph. aureus. It inhibited the above process by 93.0 and 98.8 per cent respectively. The data on the cells of Staph. aureus, as well as the data on comparison of the inhibitory effect of rifampicin derivatives with respect to the RNA-polymerase reaction in acellular systems are presented.     

Acquisition of rifabutin resistance by a rifampicin resistant mutant of Mycobacterium tuberculosis involves an unusual spectrum of mutations and elevated frequency

Background

Mutations in a small region of the rpoB gene are responsible for most rifamycin resistance in Mycobacterium tuberculosis. In this study we have sequentially generated resistant strains to first rifampicin and then rifabutin. Portions of the rpoB gene were sequenced from 131 randomly selected mutants. Second round selection resulted in a changed frequency of specific mutations.

Methods

Mycobacterium tuberculosis (strain Mtb72) rifamycin resistant mutants were selected in vitro with either rifampicin or rifabutin. One mutant R190 (rpoB S522L) selected with rifampicin had a rifampicin MIC of 32 μg/ml but remained sensitive to rifabutin (MIC<0.8 μg/ml). This mutant was subjected to a second round of selection with rifabutin.

Results

All 105 first round resistant mutants derived from the parent strain (Mtb72) screened acquired mutations within the 81 bp rpoB hotspot. When the rifampicin resistant but rifabutin sensitive S522L mutant was subjected to a second round of selection, single additional rpoB mutations were identified in 24 (92%) of 26 second round mutants studied, but 14 (54%) of these strains contained mutations outside the 81 bp hotspot (codons 144, 146, 148, 505). Additionally, spontaneous rifabutin resistant mutants were produced at >10 times the frequency by the S522L mutant than the parent strain.

Conclusion

First round selection of mutation S522L with rifampicin increased the frequency and changed the spectrum of mutations identified after selection with rifabutin.     

Production of rifamycin SV using mutant strains of Amycolatopsis mediterranei MTCC17

Production of rifamycin SV using mutant strains of Amycolatopsis mediterranei (MTCC17) has been studied. Attempts were made to increase the productivity of rifamycin SV by ultraviolet radiation and specific screening programmes for selection of superior producers. Among 20 morphologically variant mutants of A. mediterranei MTCC17 isolated, four mutants showed increased resistance to the rifamycin SV. These selected mutant isolates increased the yield of rifamycin SV from 2000 to 3250mg/l.     

Optimization of culture medium for rifamycin SV production by<Emphasis Type="Italic">Amycolatopsis mediterranei</Emphasis> MM2 using statistical designs

In this paper, we conducted a statistical optimization of the medium components for the production of rifamycin SV byAmycolatopsis mediterranei MM2. In order to maximize the yield of rifamycin SV, a Plakett-Burman experimental design (PBD) was initially utilized in the screening of the medium components among 11 nutrients. Glycerol and yeast extract were determined to influence significantly the yield of rifamycin SV. Then, a central-composite experimental design (CCD) was utilized in order to optimize the concentrations of the screened components accqired using the PBD and to predict the mutual interactions occurring between the screened components. The predicted optimal glycerol and yeast extract concentrations were determined to be 43.8 and 9.5 g/L, respectively. At this optimum point, the predicted rifamycin SV yield was 490.5 mg/L, whereas the corresponding experimental yield was 480.3±43.8 mg/L.     

Screening of rifamycin producing marine sponge bacteria by LC-MS-MS

HPLC-MS-MS has been used for the identification and characterisation of rifamycin B and rifamycin SV in various strains of the marine sponge-derived bacterium Salinispora. Gradient elution using acetonitrile/water/ammonium acetate was used to separate the rifamycins from the matrix and negative ion-electrospray mass spectrometry was used for detection and confirmation. The presence of rifamycin in bacterial extracts was confirmed by matching retention times, parent ion spectra and the fragmentated parent ion spectra of the standard compounds and the bacterial extracts. All strains of the marine sponge bacterium Salinispora tested were found to contain rifamycin thus an alternate actinobacterial source of rifamycin was established.     

Rifamycin B oxidase from Monocillium spp., a new type of diphenol oxidase

It was found that enzyme from a microbial strain, Monocillium spp. ATCC 20621, catalyzed the oxidative reaction of rifamycin B to form rifamycin O. The identification of the reaction products suggested that the reaction proceeded by the oxidative cyclization of rifamycin B to give rifamycin O, which spontaneously hydrolyzed to rifamycin S in neutral aqueous milieu. The characteristic of the enzyme was different as compared with that of other polyphenol oxidases such as laccase. It is proposed that this new type of enzyme be classified into a subgroup EC 1.10.3.6 with a trivial name rifamycin B oxidase.     

Fluorescence quenching of serum albumin by rifamycin antibiotics and their analytical application.

In neutral medium, rifamycin antibiotics such as rifapentin (RFPT), rifampicin (RFP), rifandin (RFD) and rifamycin SV (RFSV) can bind with human serum albumin (HSA) and bovine serum albumin (BSA) to form complexes, resulting in the quenching of the intrinsic fluorescence (lambda(ex)/lambda(em) = 285/355 nm) of the BSA and HSA. The quenching intensity (DeltaF) is directly proportional to the concentration of the rifamycin antibiotics. Therefore, a new analytical method was established to determine trace rifamycin antibiotics. The method had fairly high sensitivity and the detecting limits (3sigma) for RFPT, RFP, RFD and RFSV were 0.85, 0.98, 1.83, 1.89 ng/mL, respectively, for the HSA system and 0.76, 0.89, 1.55, 1.77 ng/mL, respectively, for the BSA system. All relative standard deviations (RSDs) were <3.8%. In this work, the characteristics of the fluorescence spectra were studied and the optimum reaction conditions and influencing factors were investigated. The influence of coexisting substances was tested and the results showed that the method had good selectivity and could be applied to determine trace rifamycin antibiotics in medicine capsules and urine samples. Taking the RFSV-serum albumin system as an example, the reaction mechanisms, such as binding constants, binding sites, binding distance and the type of fluorescence quenching, were investigated.     

Effect of metal ion catalyzed oxidation of rifamycin SV on cell viability and metabolic performance of isolated rat hepatocytes

The effect of rifamycin SV on metabolic performance and cell viability was studied using isolated hepatocytes from fed, starved and glutathione (GSH) depleted rats. The relationships between GSH depletion, nutritional status of the cells, glucose metabolism, lactate dehydrogenase (LDH) leakage and malondialdehyde (MDA) production in the presence of rifamycin SV and transition metal ions was investigated. Glucose metabolism was impaired in isolated hepatocytes from both fed and starved animals, the effect is dependent on the rifamycin SV concentration and is enhanced by copper (II). Oxygen consumption by isolated hepatocytes from starved rats was also increased by copper (II) and a partial inhibition due to catalase was observed. Cellular GSH levels which decrease with increasing the rifamycin SV concentration were almost depleted in the presence of copper (II). A correlation between GSH depletion and LDH leakage was observed in fed and starved cells. Catalase induced a slight inhibition of the impairment of gluconeogenesis, GSH depletion and LDH leakage in starved hepatocytes incubated with rifamycin SV, iron (II) and copper (II) salts. Lipid peroxidation measured as MDA production by isolated hepatocytes was also augmented by rifamycin SV and copper (II), especially in hepatic cells isolated from starved and GSH depleted rats. Higher cytotoxicity was observed in isolated hepatocytes from fasted animals when compared with fed or GSH depleted animals. It seems likely that in addition to GSH level, there are other factors which may have an influence on the susceptibility of hepatic cells towards xenobiotic induced cytotoxicity.     

等离子体作用结合氧限制模型选育利福霉素SV高产菌株

利福霉素SV毒性低、疗效高、抗菌谱广,主要由地中海拟无枝酸菌发酵生产,其发酵过程属于耗氧发酵,供氧直接影响产物形成.为减少发酵过程氧限制影响,进一步提高利福霉素发酵产量,通过构建定向氧限制模型,将常温常压等离子体诱变和无水亚硫酸钠氧限制筛选模型相结合,建立了利福霉素生产菌株24孔板快速培养的高通量筛选方法,高效选育出能...     

Physico-chemical properties of DNA-dependent RNA-polymerase from Escherichia coli and its subunits

CD and UV spectroscopy were employed to study at different temperatures the conformational states of the DNA-dependent RNA polymerase core- and holo-enzymes, as well as of its alpha and beta subunits. Both core- and holo-enzyme were shown to have a higher percentage of regular structures than the separate subunits. CD and fluorescence methods were used to monitor the complex formation between rifamycin SV or its derivative, rifampicin, with the RNA polymerase from the E. coli wild and mutant (Rpo B255) types, the former enzyme being sensitive and the latter being resistant to these antibiotics. Complexation led to concomitant changes in the conformation of antibiotics and local structural rearrangements of the protein in vicinity of the binding site which comprises at least one tryptophan residue in a hydrophobic microenvironment.