Synthesis and evaluation of dual-action kanglemycin-fluoroquinolone hybrid antibiotics.

Bacterial resistance threatens the utility of currently available antibiotics. Rifampicin, a cornerstone in the treatment of persistent Gram-positive infections, is prone to the development of resistance resulting from single point mutations in the antibiotic's target, RNA polymerase. One strategy to circumvent resistance is the use of 'hybrid' antibiotics consisting of two covalently linked antibiotic entities. These compounds generally have two distinct cellular targets, reducing the probability of resistance development and potentially providing simplified pharmacological properties compared to combination therapies using the individual antibiotics. Here we evaluate a series of semi-synthetic hybrid antibiotics formed by linking kanglemycin A (Kang A), a rifampicin analog, and a collection of fluoroquinolones. Kang A is a natural product antibiotic which contains a novel dimethyl succinic acid moiety that offers a new attachment point for the synthesis of hybrid antibiotics. We compare the activity of the Kang A hybrids generated via the acid attachment point to a series of hybrids linked at the compound's naphthoquinone ring system. Several hybrids exhibit activity against bacteria resistant to Kang A via the action of the partnered antibiotic, suggesting that the Kang scaffold may provide new avenues for generating antibiotics effective against drug-resistant infections.

Chronic exposure to rifaximin causes hepatic steatosis in pregnane X receptor-humanized mice.

Rifaximin, a nonsystemic antibiotic that exhibits low gastrointestinal absorption, is a potent agonist of human pregnane X receptor (PXR), which contributes to its therapeutic efficacy in inflammatory bowel disease. To investigate the effects of long-term administration of rifaximin on the liver, PXR-humanized mice were administered rifaximin for 6 months; wild-type and Pxr-null mice were treated in parallel as controls. Histological analysis revealed time-dependent intense hepatocellular fatty degeneration and increased hepatic triglycerides in PXR-humanized mice and not in wild-type and Pxr-null mice. After long-term treatment, PXR target genes were induced in small intestine and liver, with significant up-regulation in the expression of hepatic genes related to triglyceride synthesis and lipid accumulation. However, no significant hepatic accumulation of rifaximin was found, even after 6 months of treatment, in PXR-humanized mice. Genes in the small intestine that are involved in the uptake of fatty acids and triglycerides were induced along with increased triglyceride accumulation in intestinal epithelial cells of PXR-humanized mice; this was not observed in wild-type and Pxr-null mice. These findings suggest that long-term administration of rifaximin could lead to PXR-dependent hepatocellular fatty degeneration as a result of activation of genes involved in lipid uptake, thus indicating a potential adverse effect of rifaximin on liver function after long-term exposure.

Experimental and clinical studies on Rifacinna--the new effective antituberculous drug (review).

A new rifamycin derivative 3-(4-cinnamyl-piperazinyl iminomethyl) rifamycin SV (T9) and its sodium salt (T11, Rifacinna((R))) were in vitro, in vivo, toxicologically and clinically investigated in comparison with rifampicin, rifapentine, rifabutin, rifalazil. Our experiments showed that Rifacinna exhibits excellent in vitro activity against Gram(+), Gram (-) aerobic, anaerobic pathogens and mycobacteria. Rifacinna is active against Staphylococcus, Streptococcus spp. including MRSA, with MIC90- 0.06-0.5 mg/L; against Gram(+), Gram (-) anaerobes with MIC90 0.5 - 1 mg/L; against Mycobacterium tuberculosis (MTB) with MIC90 0.062 mg/L; against MDR resistant MTB (25%-30 %) and Mycobacterium avium complex (MAC) strains with MICs 0.6-1.0 mg/L. It shows high intraphagocytic activity against MAC strains (0.06 0.125mg/L). Single daily dose 10 mg/kg provides complete erradication of mycobacteria in experimental generalized tuberculosis. Pharmacological studies established: excellent pharmacokinetic profile following single oral dose 10mg/kg Tmax 5-6 h, C(max) 5-9 mg/L, T1/2 33-34 h; low toxicity; no teratogenic and embryotoxic effects. The clinical study of Rifacinna shows higher therapeutic efficacy than Rifampicin in patients with infiltrative, disseminated and cavitary form of pulmonary tuberculosis, good tolerability and safety profile. Some of the recent patents related to the treatment of tuberculosis are discussed in this review article.

Inhibition of bile acid transport across Na+/taurocholate cotransporting polypeptide (SLC10A1) and bile salt export pump (ABCB 11)-coexpressing LLC-PK1 cells by cholestasis-inducing drugs.

Vectorial transport of bile acids across hepatocytes is a major driving force for bile flow, and bile acid retention in the liver causes hepatotoxicity. The basolateral and apical transporters for bile acids are thought to be targets of drugs that induce cholestasis. Previously, we constructed polarized LLC-PK1 cells that express both a major bile acid uptake transporter human Na+/taurocholate cotransporting polypeptide (SLC10A1) (NTCP) and the bile acid efflux transporter human bile salt export pump (ABCB 11) (BSEP) and showed that monolayers of such cells can be used to characterize vectorial transcellular transport of bile acids. In the present study, we investigated whether cholestasis-inducing drugs could inhibit bile acid transport in such cells. Because fluorescent substrates allow the development of a high-throughput screening method, we examined the transport by NTCP and BSEP of fluorescent bile acids as well as taurocholate. The aminofluorescein-tagged bile acids, chenodeoxycholylglycylamidofluorescein and cholylglycylamidofluorescein, were substrates of both NTCP and BSEP, and their basal-to-apical transport rates across coexpressing cell monolayers were 4.3 to 4.5 times those of the vector control, although smaller than for taurocholate. The well known cholestatic drugs, rifampicin, rifamycin SV, glibenclamide, and cyclosporin A, reduced the basal-to-apical transport and the apical efflux clearance of taurocholate across NTCP- and BSEP-coexpressing cell monolayers. Further analysis indicated that the drugs inhibited both NTCP and BSEP. Our study suggests that such coexpressing cells can provide a useful system for the identification of inhibitors of these two transport systems, including potential drug candidates.

Pharmacokinetics and tissue distribution of rifametane, a new 3-azinomethyl-rifamycin derivative, in several animal species.

Single and repeated dose experiments in mice, rats, dogs and monkeys are reported in this study to assess the pharmacokinetics and tissue distribution of rifametane, a new semi-synthetic rifamycin with the chemical formula 3-[(1-diethylaminoethylidene)azinomethyl]rifamycin SV (CAS 94168-98-6, SPA-S-565). All the kinetic tests were carried out in comparison with known rifamycin derivatives, as rifampicin (CAS 13292-46-1) or rifamycin SV (CAS 6998-60-3). Mice received single i.v. and oral administration of 10 mg/kg of rifametane or of rifampicin and serum samples were obtained up to 96 h after dosing. The two antibiotics showed similar peak of serum concentrations, but rifametane showed a longer half-life and higher AUC values. In an additional experiment, the tissue/serum ratio after the 10 mg/kg oral dose was lower than unity for lungs and kidneys, while the liver/serum ratio exceeded the unity at all sampling times. After 4 weeks of once weekly administration measurable serum and tissue concentrations were observed, and after twice weekly administration for the same time period some blood and tissue accumulation was seen. Rats were treated with a single intravenous injection of 20 mg/kg of rifametane or rifampicin and with single oral or i.m. administration of 60 mg/kg of rifametane or reference standards (rifampicin and rifamycin SV resp.), in two separate trials. The serum half-life of the test antibiotic after i.v. dose was 6 times longer than that of rifampicin and the serum concentrations of rifametane after oral and i.m. doses were higher and longer-lasting than those of the reference compounds. Repeated daily administrations of rifametane at three dose levels (3, 10, 30 mg/kg p.o.) for 4 weeks induced very high serum and liver concentrations. Dogs received a single oral dose of 1.25 mg/kg of rifametane or 2.5 mg/kg of rifampicin. The serum half-life of rifametane resulted 3 times longer than that of rifampicin. Remarkable serum and tissue concentrations were observed after 3-4 weeks of daily oral administration of rifametane at 3, 10, 30 mg/kg dose. Monkeys were given single oral or i.m. administration of 30 mg/kg of rifametane or reference standards (oral rifampicin and i.m. rifamycin SV). The serum concentrations after rifametane were higher and more sustained than those of reference compounds and the half-lives of the test antibiotic were about 2.5 (p.o.) to 6 times (i.m.) longer. The urine excretion of rifametane after a single intravenous dose in rats and a single oral dose in dogs was very low, while rifampicin had a little higher urine concentrations.

Isolation of anaerobic respiratory mutants of Shewannella putrefaciens and genetic analysis of mutants deficient in anaerobic growth on Fe3+.

A genetic approach was used to study (dissimilatory) ferric iron (Fe3+) reduction in Shewanella putrefaciens 200. Chemical mutagenesis procedures and two rapid plate assays were developed to facilitate the screening of Fe3+ reduction-deficient mutants. Sixty-two putative Fe3+ reduction-deficient mutants were identified, and each was subsequently tested for its ability to grow anaerobically on various compounds as sole terminal electron acceptors, including Fe3+, nitrate (NO3-), nitrite (NO2-), manganese oxide (Mn4+), sulfite (SO3(2-)), thiosulfate (S2O3(2-)), trimethylamine N-oxide, and fumarate. A broad spectrum of mutants deficient in anaerobic growth on one or more electron acceptors was identified. Nine of the 62 mutants (designated Fer mutants) were deficient only in anaerobic growth on Fe3+ and retained the ability to grow on all other electron acceptors. These results suggest that S. putrefaciens expresses at least one terminal Fe3+ reductase that is distinct from other terminal reductases coupled to anaerobic growth. The nine Fer mutants were conjugally mated with an S. putrefaciens genomic library harbored in Escherichia coli S17-1. Complemented S. putrefaciens transconjugants were identified by the acquired ability to grow anaerobically on Fe3+ as the sole terminal electron acceptor. All recombinant cosmids that conferred the Fer+ phenotype appeared to carry a common internal region.

[Preclinical research on drug forms containing rifamycin SV intended for intramammary use]. / Predklinichni izsledvaniia vurkhu lekarstveni formi, sudurzhashti rifamitsin SV, prednaznacheni za intramamarna upotreba.

It was established, that LD50 of rifamycin SV--sodium salt, produced by the Research Institute on Microbiological Industry--Razgrad (People's Republic of Bulgaria)--RM, at p.o. applying on albino mice, albino rats and broilers is correspondingly greater than 4100.0 mg/kg m., greater than 5740.0 mg/kg m. and 1951.6 (1355.5 divided by 2810.1) mg/kg m. and at s. c. introducing into albino mice--815.9 (756.9 divided by 879.9) mg/kg m. From the prepared on the basis of RM-4 intramammary medicaments--RM-1, RM-2 and RM-3 for lactating cows and RM-4 for cows in the dry period, a good bearing have RM-1, RM-3 and RM-4. RM-1, RM-2 and RM-3, applied intracisternally a single time on lactating cows, and RM-4 on cows in the dry period, create antimicrobial levels with duration correspondingly 168, 72 and 48 h, and 35 days. RM almost does not penetrate in the neighbouring not treated quarters. Appropriate medicament for the lactating cows with average duration of emission is RM-3 (term of action 96 h) and for cows in the dry period RM-4 when applied 35 days before the calving.

Acute, subacute, chronic toxicity and mutagenicity studies of rifaximin (L/105) in rats.

Acute, subacute and chronic toxicity-mutagenicity studies of rifaximin (L/105), a new semisynthetic rifamycin SV antibiotic, were conducted in rats. The oral LD50 value for this species was greater than 2000 mg/kg. Rifaximin given orally to rats up to 6 months produced no evident adverse effects up to 100 mg/kg. The moderate effects noted were probably due to the topical action of the drug. Rifaximin did not show any mutagenic activity when compared with mutagenic standards.

Fertility study of rifaximin (L/105) in rats.

The influence of rifaximin (L/105) on conception and pregnancy in rats up to F1 progeny was reported. Rifaximin showed a good fertility rate and no dose-related differences in the number of viable or dead fetuses per litter (Fo progeny) up to 100 mg/kg. The number of live or dead pups at birth, pup weight, gestation length and survival of offspring to weaning were not influenced up to the highest dose, 100 mg/kg (Fo-F1 progeny).

[Teratogenic action of Rifaximin in the rat and rabbit and its effect on perinatal development in the rat]. / Azione teratogena del Rifaximin nel ratto e nel coniglio e sua influenza sullo sviluppo perinatale del ratto.

The purpose of this study is to evaluate the teratogenic action and the effect on perinatal development of Rifaximin. Teratogenic action of Rifaximin was studied in the rat and in the rabbit. The drug was administered by gavage during organogenesis period at 50 and 100 mg/kg; idroxyurea and thalidomide were used as teratogenic drugs. The effect on physical and behavioural development of the pups was studied in the rat at 50 and 100 mg/kg during the foetal period and suckling time. The search for malformation of internal organs was carried out on the half of the fetuses fixed according to Wilson; the examination of the skeleton of the other fetuses was carried out according to Dawson. Some parameters of physical and behavioural development were evaluated in the pups. Rifaximin at these dosages has no teratogenic effects and does not influence the psychophysical development of the pups.

Change in the surface hydrophobicity of substrate cells during bdelloplast formation by Bdellovibrio bacteriovorus 109J.

During intraperiplasmic growth of Bdellovibrio bacteriovorus 109J, the substrate cell surface becomes more hydrophobic. This was shown (i) by comparing the sensitivity to hydrophobic antibiotics of wild-type and lipopolysaccharide mutant strains of Salmonella typhimurium to that of the bdellovibrio growing on these strains and (ii) by measuring the binding efficiency of these strains, Escherichia coli, and their derived bdelloplasts to octyl Sepharose. The kinetics of increase in surface hydrophobicity was similar to the kinetics of the conversion of the substrate cell peptidoglycan to a lysozyme-resistant form (M. Thomashow and S. Rittenberg, J. Bacteriol. 135:1008-1014, 1978), and hydrophobicity reached a maximum at about 60 min in a synchronous culture. The change in hydrophobicity was inhibited by chloramphenicol, suggesting that bdellovibrio protein synthesis was required. Control experiments revealed that the free-swimming bdellovibrio had a more hydrophobic surface than the deep rough mutants of S. typhimurium.

MIC values do not predict the intraphagocytic killing of Staphylococcus aureus by naphthalenic ansamycins.

Ten naphthalenic ansamycins were compared for their ability to kill extracellular or phagocytosed Staphylococcus aureus 502A. These included rifamycins, streptovaricins and tolypomycin Y. Although the compounds differed markedly in killing extracellular S. aureus, there was surprisingly little difference between them in assisting human leukocytes to kill phagocytosed S. aureus. In fact, when compared to rifampin, some ansamycins that were less effective in killing extracellular bacteria were more effective in killing phagocytosed bacteria. These data, together with an analysis of structure and activity, suggested that a specific transport mechanism might be involved. First considered was a vitamin K transport mechanism. Indeed warfarin, a vitamin K antagonist, blocked the ability of rifampin to kill phagocytosed S. aureus, as did the coumarins, novobiocin and coumarin-3-carboxylic acid. However, direct evidence for a vitamin K transport mechanism could not be obtained using vitamin K preparations. The fused phenolic, bicyclic system common to all of these ansamycins was tentatively considered to be the portion necessary for phagocyte penetration.

LM 427, a new spiropiperidylrifamycin: in vitro and in vivo studies.

The spiropiperidylrifamycin LM 427 (4-deoxo-3,4-[2-spiro-N-isobutyl-4-piperidyl]-(1H)-imidazo-(2,5-dihydro) rifamycin S) displays a broad spectrum of potent antibacterial activity in vitro. In vivo it is particularly effective in the therapy of experimental tubercular infections of mice. Three schedules of treatment were employed and the best results were obtained when intermittent administrations were used (ED50 of LM 427; 7 times lower than rifampicin). LM 427 is well distributed in tissues of mice and rats, with lung concentrations 10 to 20 times higher than plasma levels.

Novel rifamycins. III. Synthesis and antibacterial activity of 3-amidino- and of 4-aminoimidazolo[4,5-c]rifamycin derivatives.

A number of semisynthetic rifamycin derivatives modified at position 3 and/or 4, belonging to general structures 2 and 4 (see Scheme 1), have been obtained. The synthesis and the biological activities of the new compounds are described. Compounds 4p and 4q display very good antimycobacterial activity in mice.

The quantitative structure-activity relationship of rifamycin B amide and hydrazide toxicity in mice.

Quantitative structure-activity relationships (QSAR) are developed for the acute toxicity of a number of rifamycin B amides and hydrazides in mice. The equations indicate a parabolic dependence of toxicity on the partition coefficient. Two related compounds, rifamycin SV and rifamycin B, were included with the amides in a more general correlation. The latter equation was used to predict the toxicity of rifampicin, suggesting that this relationship may be of general use for the prediction of rifamycin toxicity.

Effect of the rifamycin dimers on the activities of nucleic acid polymerases from various sources. Relation between lipophily and toxicity.

The chemical dimers of rifamycin SV resembled the corresponding monomeric analogs with respect to the inhibitory properties versus the nucleic acid polymerases. At low doses, such compounds blocked the initiation step of the DNA transcription catalyzed by the bacterial RNA polymerase, as observed for the parental antibiotic and its derivative rifampicin which are largely used in therapy. At concentrations one to two orders of magnitude higher, the chemically modified rifamycins inhibited also other nucleotidyltransferases. The widespread toxicity of the dimeric and monomeric semisynthetic rifamycins versus these enzymes was not causally related with an enhancement of their lipophily. The observed effects might be due to a loss of selectivity in the inhibition mechanism which was originally specific for the RNA polymerase from E. coli at the beginning of its catalysis. The rifamycin derivatives might then react with the catalytic portion of other nucleotidyltransferases interfering adversely with the enzyme activity in a number of ways and/or at different levels.