Synthesis and spectroscopic and electrochemical characterisation of a conducting polythiophene bearing a chiral beta-substituent: polymerisation of (+)-4,4 bi.

A regioregular head-to-head/ tail-to-tail poly(beta,beta'-disubstituted bithiophene) P1 was synthesised by chemical and electrochemical polymerisation of 2,2'-bithiophene that bears (S)-2-methylbutylsulfanyl chains in the beta and beta'-positions. The polymer was characterised by GPC, NMR and UV/Vis spectroscopy, CD, AFM and by electrochemical and conductivity measurements. The CD spectra of P1 in solutions in which poor solvents are present show interesting features and allow the presence of different optically active species to be distinguished. Upon varying the casting conditions of P1, different relative amounts of grainy and homogeneous aggregated phases were observed in AFM micrographies of films and corresponding negative or positive first Cotton effects were found in the CD spectra. AFM, CD and UV/Vis characterisations were also performed on an electrogenerated optically active polymer PE1, in order to make a comparison with the chemically synthesised one. The interesting, small band gap of P1 allows for easy p- and n-electrochemical doping.

Teicoplanin Chemistry and Microbiology.

The chemistry, microbiology and mode of action of teicoplanin, as well as the mechanism, control and epidemiology of glycopeptide resistance, are discussed in detail. The antibacterial activity of teicoplanin against Gram-positive bacteria, including those expressing resistance to unrelated compounds, is similar to that of vancomycin but with increased potency, particularly against Streptococcus spp and Enterococcus spp. Some strains of coagulase-negative Staphylococcus spp, particularly S. haemolytieus, are less susceptible to teicoplanin than to vancomycin. Teicoplanin is active against vancomycin resistance caused by VanB and VanC, but is not active against VanA resistant strains. The epidemiology of GISA and VISA strains of S. aureus is, as yet, poorly understood with more work necessary to elucidate the sequence of events leading to their evolution. Despite the increasing importance of glycopeptide resistance, teicoplanin has proved its clinical worth and continues to have important potential in the treatment of life-threatening Gram-positive sepsis.

In-vitro and in-vivo antibacterial activity of BI 397, a new semi-synthetic glycopeptide antibiotic.

BI 397 (formerly A-A-1) is a semisynthetic derivative of the teicoplanin-like glycopeptide A40926. It was more active in vitro against staphylococci (including some teicoplanin-resistant strains) than teicoplanin and vancomycin. Against streptococci (including penicillin-resistant strains) BI 397 has activity comparable with that of teicoplanin and better than vancomycin. BI 397, when administered to rats by the i.v. route, gives high and long lasting blood levels. It shows excellent activity in models of acute septicaemia in immunocompetent and neutropenic mice. In a rat staphylococcal endocarditis model it is as effective as teicoplanin and vancomycin at reducing bacterial loads in the heart, but at lower dosages and with a reduced number of daily treatments compared with the two glycopeptide controls. BI 397 is highly efficacious in clearing penicillin-susceptible and -resistant pneumococci from lungs of immunocompetent and neutropenic rats. The data from these studies show that BI 397 combines an excellent in-vitro antibacterial activity with favourable pharmacokinetic behaviour resulting in potent in-vivo activity.

Diffusion of teicoplanin and vancomycin in agar.

Teicoplanin, although more active than vancomycin [by minimum inhibitory concentration (MIC)], produces smaller inhibition zones in sensitivity testing with 30-microgram disks. Our data support the hypothesis that this is due to lower diffusion of teicoplanin in agar media. After 6 hr of incubation, approximately 70% of vancomycin, but only 20% of teicoplanin entered the agar from a paper disk charged with 30 micrograms of antibiotic. This is due to a difference between the diffusion coefficients: 0.47 mm2/hr for teicoplanin and 0.72 mm2/hr for vancomycin. With the methodology used in this work, it is possible to calculate the range of concentrations of the antibiotic occurring at times likely to include the critical time--the time when the inhibition zone is formed--of most strains at any given distance from the reservoir. One can thus estimate the breakpoint diameter for a given MIC breakpoint; for example, an MIC breakpoint of less than or equal to 4 micrograms/ml would correspond to a greater than or equal to 15-mm breakpoint diameter for vancomycin (30-microgram disk) and a greater than or equal to 13-mm breakpoint diameter for teicoplanin (30-microgram disk).

In-vitro activity of vancomycin, teicoplanin, daptomycin, ramoplanin, MDL 62873 and other agents against staphylococci, enterococci and Clostridium difficile.

The minimum inhibitory concentrations (MICs) of different antibiotics were determined by a broth microdilution method for staphylococci, enterococci and Clostridium difficile. The antimicrobial agents tested were vancomycin, teicoplanin, daptomycin, ramoplanin, MDL 62873, rifampicin and piperacillin, the latter limited to enterococci. In terms of MIC90S, daptomycin (0.89 mg/l). MDL 62873 (0.99 mg/l), and teicoplanin (1.50 mg/l) were found to be highly active against methicillin-resistant Staphylococcus aureus (MRSA). Daptomycin (MIC90 0.48 mg/l), MDL 62873 (0.95 mg/l) and ramoplanin (1.45 mg/l) were the most active drugs against methicillin-resistant S. epidermidis (MRSE). Teicoplanin (MIC90 0.45 mg/l) was the most active agent against enterococci, followed by MDL 62873 (0.65 mg/l) and daptomycin (1.60 mg/l). MDL 62873 gave the lowest MIC90 (0.17 mg/l) for C. difficile. Teicoplanin (MIC90 0.42 mg/l), daptomycin (0.87 mg/l) and ramoplanin (0.98 mg/l) were also very active. Our results indicate that teicoplanin, daptomycin, ramoplanin and MDL 62873, a teicoplanin derivative, are potentially effective alternative antibiotics for treatment of infections caused by staphylococci, enterococci and C. difficile.

Synthesis and biological activity of some derivatives of rifamycin P.

A series of derivatives of rifamycin P, an antibiotic produced by fermentation of a mutant strain of Nocardia mediterranea or by chemical modification of rifamycin S, have been prepared. The structures of these compounds were determined by 1H NMR, IR, UV, and LC/MS. Their in vitro and in vivo antibacterial activities in comparison with rifampicin and two other rifamycins under investigation were evaluated. The derivatives were more active than rifamycin P against Mycobacterium avium complex and other slowly and rapidly growing nontuberculous mycobacteria which frequently cause systemic infection in patients with AIDS. 2'-(Diethylamino)rifamycin P (P/DEA) appears suitable for further investigation.

Ramoplanin: a review of its discovery and its chemistry.

Ramoplanin is a novel antibacterial agent with characteristics that make it suitable for development as a topical treatment for acne, infected wounds, and for treatment of antibiotic-associated diarrhoea. In this paper, the authors will review the discovery process and the descriptive chemistry of this novel molecular entity.

The differential glycosylation of human pro-urokinase from various recombinant mammalian cell lines does not affect activity and binding to PAI-1.

Human chromosomal DNA encoding single-chain urokinase-type Plasminogen Activator (scu-PA, or pro-urokinase) was inserted in an expression plasmid and transfected in human A431, mouse LB6 and CHO cells. LB6 cells were also transfected with a Bovine Papilloma Virus derivative containing the scu-PA gene. Human scu-PA was purified from cell supernatants of recombinant clones and characterized for structure and function. All recombinant scu-PAs are undistinguishable from human urine-derived scu-PA for peptide backbone, but possess a higher sugar content, as revealed by SDS-PAGE analysis after digestion with glycopeptidase F. This difference is partly due to an increased sialic acid content, as shown by analysis of neuraminidase-treated scu-PAs. No difference was found, however, among recombinant and natural scu-PAs in the kinetics of conversion into two-chain active forms (tcu-PAs) by human plasmin, and in the KM and kcat values of tcu-PA activity on the chromogenic substrate S-2444 and on human plasminogen. Also, recombinant and non-recombinant tcu-PAs displayed similar dose-response curves for binding to the endothelial inhibitor PAI-1. In conclusion, the glycosylation pattern of u-PA does not affect its interaction with the plasma proteins directly involved in its fibrinolytic function.

Epitope mapping of the anti-urokinase monoclonal antibody 5B4 by isolated domains of urokinase.

The amino terminal fragment (ATF) of urokinase-type plasminogen activator (uPA) is a degradation product comprising the entire growth factor-like and kringle domains. It has been previously shown that ATF is able to bind to the u-PA receptor through the growth factor-like domain and that the anti u-PA monoclonal antibody 5B4 (Mab 5B4) binds to ATF preventing u-PA receptor binding. To localize more precisely the epitope recognized by Mab 5B4, ATF was subfragmented by controlled enzymatic proteolysis with V8 protease. Three subfragments of 4,000 Mr (F-4k), 11,000 Mr (F-11k) and 12,000 Mr (F-12k) were purified from the reaction mixture and characterized. SDS-PAGE under reducing and non-reducing conditions, N-terminal aminoacid sequence analysis and C-terminal aminoacid analysis of each fragment indicate that F-4k and F-11k correspond to intact growth factor-like domain and kringle domain (residues 4-43 and 44-135 respectively) while F-12k corresponds to the kringle domain cleaved in the first loop at the glu52-gly53 bond. By Western blot and competitive binding experiments we show that Mab 5B4 recognizes an epitope located on the kringle domain of u-PA and that the binding is strongly reduced when the kringle contains an additional cleavage in its first loop. Since the receptor binding site of u-PA has been previously shown to be located on the growth factor-like domain, Mab 5B4 inhibits the binding of uPA to its cellular receptor likely by steric hindrance. Besides the proven utility in epitope localization of anti u-PA monoclonal antibodies, these u-PA fragments may represent powerful tools for studies of structure-function relationship of u-PA.

Cloning of a DNA region of Actinoplanes teichomyceticus conferring teicoplanin resistance.

Teicoplanin is a glycopeptide antibiotic, produced by Actinoplanes teichomyceticus, active against Gram positive bacteria and recently introduced into clinical practice. It blocks cell wall biosynthesis by inhibiting peptidoglycan polymerization. The mechanism(s) of resistance of the producer strains of this class of antibiotics have not yet been characterized. We have constructed a genomic bank of A. teichomyceticus in Streptomyces lividans. A clone from this bank, PTR168, was able to confer resistance to teicoplanin on its sensitive host. The restriction map of plasmid pTR168 and the hybridization pattern to A. teichomyceticus DNA were determined; we have also studied the mechanism of this resistance which seems correlated with a reduced binding of the antibiotic to the cell wall.

A42867, a novel glycopeptide antibiotic.

A42867 is a new glycopeptide antibiotic of the ristocetin-vancomycin class active against aerobic and anaerobic Gram-positive bacteria. A42867 is produced by a strain of Nocardia nov. sp. ATCC 53492. A42867 was isolated during a screening program aimed at the discovery of new members of this glycopeptide class of antibiotics, by affinity chromatography based on an acyl-D-alanyl-D-alanine probe. The structure of A42867 was elucidated by fast atom bombardment MS, high field 2D 1H NMR spectroscopy, and HPLC analysis of the hydrolyzed carbohydrates. A42867 differs from vancomycin in the sugar portion and in the presence of only one chlorine atom in the peptide core. Its biological activity on Gram-positive aerobic and anaerobic bacteria is similar to that of other antibiotics of this group.

New experimental drugs for the treatment of tuberculosis.

New antitubercular agents are needed for two main purposes: to further simplify therapy (through reductions in the number of medicaments used, the number of doses administered, and the duration of treatment required), thus facilitating supervision and improving compliance, and to combat resistant mycobacteria. Reduction in the number of medicaments has been achieved by combining two or more drugs in a single tablet while retaining a degree of bioavailability similar to that of the single components. The adverse effects observed with once-weekly high doses of rifampin have limited the development of widely spaced intermittent regimens of treatment. For this reason new rifamycins have been developed that are as active as rifampin against mycobacteria but that also offer the advantage of high and prolonged serum levels and thus have the potential for once-weekly administration. The in vitro and in vivo properties of these drugs have been studied. Three classes of drugs show promise for the treatment of drug-resistant tuberculosis: spiropiperidyl rifamycin, the fluoroquinolones, and combinations of beta-lactam agents and beta-lactamase inhibitors.

Glycopeptide antibiotics.

Numerous glycopeptides continue to be described in the literature. They all share a similar heptapeptidic structure with a fixed spatial configuration that forms the basis of their ability to recognize D-alanyl-D-alanine-containing structures in the cell wall. This complexation results in block of peptiglycan elongation; hence, inhibition of growth; and, eventually, cell death. The great variety of substituents on the heptapeptide forms the basis of a wide gradation of physico-chemical characteristics, namely net charge and lipophilicity, which, in turn, might explain the widely differing pharmacologic properties.

A multicentre open clinical trial of teicoplanin in infections caused by gram-positive bacteria.

A multicentre open trial of teicoplanin in 81 centres in nine European countries included 1431 cases: 531 female, 900 male; mean age 49.4 years, range 1-93 years. These were hospitalized patients most of whom had infections caused by Staphylococcus aureus (816 isolates). Of a total of 1427 Gram-positive pathogens 280 (19.6%) were methicillin resistant. There were 536 skin and soft tissue infections, 263 septicaemias, 135 lower respiratory tract infections, 179 joint and bone infections and 83 endocarditides. More than a third of the infections were severe. Complicating medical factors were present in 69% of cases, including malignant disease in 14% and diabetes mellitus in 11%. Mean teicoplanin dose was 289 mg/day; mean duration of treatment was 14 days. A total of 471 patients received a high dose regimen, 400 mg teicoplanin daily for at least five days. Monotherapy with teicoplanin was used in 1037 cases and combination with other antibiotics in 394. Overall 91.7% of the 1333 evaluable cases were clinical cured or improved. The MIC of teicoplanin was less than or equal to 1 mg/l for 90% of Gram-positive isolates. Adverse events were reported in 189 cases (13.2%). The most common drug-related event was an allergic type skin reaction which occurred in 35 cases (2.4%). Transient hepatic dysfunction was reported in 28 patients (2.0%).

Structure and mechanism of action of teicoplanin.

The chemistry, structure and physicochemical properties of the novel glycopeptide teicoplanin are reviewed. The possible relationship between these characteristics and the mechanism and mode of action and pharmacokinetic behaviour are briefly discussed.