Cephalosporins to carbapenems: 1-oxygenated carbapenems and carbapenams.

The photo "Wolff" rearrangement of readily available 2-diazoceph-3-em oxides (1) directly affords carbapen-2-ems, allowing a facile entry into a ring system previously accessible only by total synthesis, lengthly semisynthesis or fermentation. The chirality of the cephalosporin is accurately translated into the corresponding carbapenem. The resulting 1-oxocarbapenems (2) were selectively transformed through reduction into 1-oxygenated carbapenems and carbapenams (3 and 4, respectively). On microbiological screening, a carbapenem (3c) was found to possess a broad spectrum of activity. An interesting antibacterial profile was discovered for a carbapenam (26).

Ring contraction of oleandrose on the macrolide antibiotic oleandomycin with [(Methoxycarbonyl)sulfamoyl]triethylammonium hydroxide inner salt.

Ring contraction of the neutral oleandrose sugar in the 14-membered-ring macrolide antibiotic oleandomycin (2) has been accomplished using [(methoxycarbonyl)sulfamoyl]triethylammonium hydroxide inner salt (1). The product of this interesting rearrangement, after methanolic hydrolysis of the 2'-acetate, is the 11-acetyl-3-O-(3"-methoxy-4"-vinylfuranosyl)oleandomycin (12). The in vitro activity of furanoside 12 is only moderately less than that of 11-acetyloleandomycin (13).

Susceptibility and resistance emergence studies with macrolides.

An extended elimination half-life and good tissue penetration enable oral azithromycin to attain high and prolonged concentrations in infected tissues, yielding high antibacterial activity in vivo. It has been suggested, however, that prolonged subinhibitory concentrations of azithromycin from 2 to 4 weeks after therapy may lead to the emergence of azithromycin resistance in vivo, compared with other macrolide antibiotics. Data from two types of in vitro susceptibility studies, an animal tissue infection model, and a clinical pediatric study demonstrate that prolonged tissue concentrations of azithromycin are unlikely to lead to the emergence of resistance in the clinical setting. Further, data from in vitro susceptibility studies indicate that resistance to macrolides, and azithromycin in particular, is significantly over-estimated for bacterial strains incubated in the presence of CO2.

(6R,8S)-(2-benzimidazolyl)hydroxymethylpenicillanic acids as potent antibacterial agents and beta-lactamase inhibitors.

(6R,8S)-(2-Benzimidazolyl)hydroxymethylpenicillanic acids (1a-1x) are potent antibacterial agents and beta-lactamase inhibitors against Gram-positive bacteria and Haemophilus influenzae. The corresponding (6R,8R)-isomers (2a-2x), the 6,6-spiro benzimidazole-penam alcohol (3), (7R,9S)-(2-benzimidazolyl)hydroxymethylcephalosporanic acid (4), and 6 beta-(2-benzimidazolyl)aminopenicillanic acid (5) are much less active as antibacterials or beta-lactamase inhibitors. The syntheses and structure-activity relationships of these compounds are discussed. Antibacterial activity and beta-lactamase inhibition data are presented.

Structure-activity relationships of 6-(heterocyclyl)-methylene penam sulfones; a new class of beta-lactamase inhibitors.

6-(Heterocyclyl)methylene penam sulfones (1) are effective beta-lactamase inhibitors and potent ampicillin and cefazolin potentiators against both Gram-positive and Gram-negative beta-lactamase producing bacteria. Several of these analogs having a pi-deficient 2-heteroaryl substituent attached to the C6-methylene position showed better inhibitory activity than clavulanic acid, Ro 15-1903, 6 beta-bromopenicillanic acid, and sulbactam against a variety of beta-lactamases. The compounds were devoid of any antibacterial activity, but in combination with ampicillin or cefazolin, exhibited synergistic activity at least equal to clavulanic acid, Ro 15-1903, 6 beta-bromopenicillanic acid or sulbactam against beta-lactamase producing strains. Structure-activity relationships for a number of compounds are described. The structure-activity relationships can be rationalized by an enzyme inhibition mechanism which we have previously proposed on the basis of methanolysis of 6-(2-pyridyl)methylene penam sulfone (1a). Two synthetic routes to prepare compounds of structural type 1 via either a Wittig reaction or an aldol condensation are reported. beta-Lactamase inhibition and MIC data are presented.

In vitro microbiological characterization of novel cyclic homopentapeptides, CP-101,680 and CP-163,234, for animal health use.

Two cyclic homopentapeptides, CP-101,680 and CP-163,234 [6a-(3',4'-dichlorophenylamino) analogs of viomycin and capreomycin, respectively], were identified as novel antibacterial agents for the treatment of animal disease, especially for livestock respiratory disease. The in vitro microbiological characterization of both CP-101,680 and CP-163,234 was carried out using their parent compounds, viomycin and capreomycin, as controls. This characterization included antibacterial spectrum, influence of media, inoculum size, pH, EDTA, polymixin B nonapeptide (PMBN), serum, cell-free protein synthesis inhibition, and time-kill kinetics. Our results indicated that the capreomycin analog, CP-163,234, showed slightly improved in vitro potency over the viomycin analog, CP-101,680. Both analogs showed very potent cell-free protein synthesis inhibition activity and were bactericidal against Pasteurella haemolytica, P. multocida and Actinobacillus pleuropneumoniae at the level of 4 times and 8 times MICs. CP-163,234 was bactericidal at the level of 4x and 8x MIC against E. coli, but re-growth was observed after 24 hours incubation at both concentrations of CP-101,680.

In vitro microbiological characterization of novel macrolide CP-163,505 for animal health specific use.

A novel 16-membered-ring macrolide agent (CP-163,505, a reductive amination derivative of repromicin) was identified as an antibacterial against Pasteurella haemolytica, P. multocida and Actinobacillus pleuropneumoniae, important etiological agents of livestock respiratory disease. In vitro MIC50/90 analysis revealed that CP-163,505 was more potent (4x) than tilmicosin against P. multocida, and equivalent to tilmicosin against P. haemolytica and A. pleuropneumoniae. In time kill kinetic studies, CP-163,505 showed bactericidal activity against P. haemolytica, P. multocida and A. pleuropneumoniae and bacteriostatic activity against E. coli at 8 times its MIC. In vitro, CP-163,505 was more potent in alkaline pH (16 approximately 32 x ) and less potent in the presence of excess cations (Mg+2 and Ca+2, 4x). EDTA and PMBN increased CP-163,505 potency against E. coli (4x) but not against the other species. Similar results were obtained with erythromycin A and tilmicosin, which were used as controls. From our data, we hypothesize that Pasteurella and Actinobacillus have an outer membrane significantly different from that of the typical enteric Gram-negative bacterium E. coli.

Correlation between the binding of beta-lactam antibiotics to Staphylococcus aureus and their physical-chemical properties.

The rate of (14)C-benzylpenicillin (penicillin G) binding to Staphylococcus aureus Oxford cells increased with increasing hydrogen ion concentration. The extent of inhibition of (14)C-penicillin G binding caused by a competing (12)C-beta-lactam antibiotic is a function of hydrogen ion concentration and can be correlated both with net charge of a competing (12)C-molecule and net charge of the S. aureus cell at a given pH. The ability of a beta-lactam antibiotic to compete for (14)C-penicillin G-binding sites can generally be correlated with its hydrophobic nature. It is proposed that, although semisynthetic cephalosporins are chemically less reactive than penicillins, they are superior to benzylpenicillin in their ability to permeate the outer surface of the Staphylococcus cell wall and irreversibly bind to reactive sites.

Comparison of the effects of the new azalide antibiotic, azithromycin, and erythromycin estolate on rat liver cytochrome P-450.

Erythromycin and some other macrolide antibiotics can first induce a cytochrome P-450 isozyme similar to the one induced in rats by pregnenolone-16 alpha-carbonitrile and then inhibit it by forming a stable cytochrome P-450-metabolite complex. The purpose of this study was to compare azithromycin, a novel 15-membered ring azalide, and erythromycin estolate for the potential to cause hepatic microsomal enzyme induction and inhibition in Sprague-Dawley rats. The daily oral administration of 800 mg of erythromycin estolate per kg for 7 days resulted in statistically significant elevations of NADPH-cytochrome c reductase, erythromycin N-demethylase (3.2-fold), and total cytochrome P-450 content. Approximately 40% of cytochrome P-450 was complexed with erythromycin metabolite. In contrast, the daily administration of 200 mg of azithromycin per kg for 7 days caused significant elevations of N-demethylase (2.5-fold) only and did not produce any increases in total cytochrome P-450 content or NADPH-cytochrome c reductase. No complexed cytochrome P-450 was detected in the azithromycin-dosed rats despite liver concentrations of azithromycin that were 118 times greater than the liver concentrations of erythromycin estolate in erythromycin estolate-dosed rats. Although the short-term oral administration of azithromycin produced hepatic accumulation of the drug and elevated azithromycin demethylase activity, there was no other evidence of hepatic cytochrome P-450 induction or inactivation via cytochrome-metabolite complex formation. In contrast to erythromycin estolate, azithromycin is not expected to inhibit its own metabolism or that of other drugs via this pathway.

CP-45,899 in combination with penicillin or ampicillin against penicillin-resistant Staphylococcus, Haemophilus influenzae, and Bacteroides.

CP-45,899 is a new, semisynthetic beta-lactamase inhibitor. When tested alone, CP-45,899 displayed only weak antibacterial activity, with the notable exception of its potent action against penicillin-susceptible and -resistant Neisseria gonorrhoeae. A combination of 3.12 microgram of CP-45,899 per ml with 3.12 microgram of ampicillin per ml, tested in broth cultures, inhibited ca. 90% of resistant Staphylococcus and Haemophilus influenzae strains; similar data were obtained in a variety of media. The same combination of CP-45,899 with ampicillin or penicillin G inhibited 90% of Bacteroides fragilis as interpreted from agar dilution minimal inhibitory concentrations. Inhibitory concentrations of CP-45,899-ampicillin were bactericidal against H. influenzae strains and were as bactericidal as nafcillin or cephalothin against S. aureus. Ampicillin-resistant S. aureus, H. influenzae, and B. fragilis strains did not develop resistance to CP-45,899-ampicillin when transferred as many as six passages in the presence of a sublethal concentration of the combination.

Laboratory studies with a new broad-spectrum penicillin, pirbenicillin.

Pirbenicillin {6-[d-2-phenyl-2(N-4-pyridylformimidoylaminoacetamido) -acetamido]-penicillanic acid} showed broad-spectrum antibacterial activity in vitro and also in the treatment of experimental infections after parenteral administration to mice. Against Pseudomonas aeruginosa, a three- to fourfold potency advantage over carbenicillin was seen both in vitro and in vivo. The in vitro antibacterial spectrum of pirbenicillin includes Escherichia coli, Serratia, Citrobacter, and Enterobacter isolates, against which it exhibited minimal inhibitory concentration values comparable to those of carbenicillin. However, mice infected with E. coli and Serratia were protected at doses of pirbenicillin that were two to four times lower than those required of carbenicillin. Pirbenicillin was more active than carbenicillin against gram-positive bacteria, especially Streptococcus faecalis. It was less active than carbenicillin against Proteus spp. and was inactive against ampicillin-resistant E. coli strains. Pirbenicillin was bactericidal at concentrations generally equal to or only two-fold higher than the minimal inhibitory concentration. With appropriately buffered media, pirbenicillin demonstrated eight- and fourfold better minimal bactericidal concentration values towards Pseudomonas isolates than those of carbenicillin and ticarcillin, respectively.

Pirbenicillin: pharmacokinetic parameters in mice.

The rapid intravenous administration to mice of pirbenicillin, carbenicillin, and ampicillin produced biexponential blood concentration-time curves when assessed by frequent blood samplings at short intervals. The pharmacokinetic behavior of pirbenicillin and the other penicillins was analyzed by the two-compartment open model. This is thought to be the first study giving detailed pharmacokinetic values of penicillins in mice. Some significant differences were noted between the pharmacokinetic values of pirbenicillin, ampicillin, and carbenicillin. These values suggest that the interchange of pirbenicillin between the central and peripheral body compartments of the mouse was slower than that of either carbenicillin or ampicillin and indicated that a greater fraction of the pirbenicillin than the ampicillin dose reached the peripheral compartment.

Laboratory evaluation of 3-(5-tetrazolyl) penam, a new semisynthetic beta-lactam antibacterial agent with extended broad-spectrum activity.

In the new agent 3-(5-tetrazolyl)penam, hereafter referred to as CP-35,587, the carboxyl function at C3 in the penicillin nucleus has been replaced with the 5-tetrazolyl moiety. Marked changes in spectrum and resistance to gram-negative beta-lactamases, particularly with regard to Klebsiella pneumoniae isolates, were conferred by this modification. The anti-Klebsiella activity clearly distinguishes the antibacterial spectrum of CP-35,587 from any known broad-spectrum penicillin. Compared to orally active cephalosporins, the spectrum advantage of CP-35,587 encompasses Enterobacter, Serratia marcescens, Citrobacter, Providencia, Haemophilus influenzae, and Streptococcus faecalis, both in vitro and in murine infections produced by many of the above-named microorganisms. Thus, CP-35,587 combines and extends the antibacterial activity of broad-spectrum penicillins and orally active cephalosporins.

Correlation of the extravascular pharmacokinetics of azithromycin with in-vivo efficacy in models of localized infection.

Infection models were used to clarify the roles of serum and extravascular concentrations in the in-vivo efficacy observed with azithromycin. In-vivo experiments were designed to give serum concentrations well below the MIC and tissue levels generally above the MIC at time of challenge and during the course of infection. The efficacy of azithromycin against a Salmonella enteritidis oral challenge (a tissue-associated infection model) in mice correlated directly with azithromycin liver levels, but not serum concentrations. The significance of extravascular pharmacokinetics was observed in a comparative study of azithromycin and ciprofloxacin against the salmonella challenge. Ciprofloxacin has a greater than 100-fold in-vitro potency advantage over azithromycin against this organism, but azithromycin (5 mg/kg) produced a greater reduction in cfu than ciprofloxacin (100 mg/kg) at the primary site of infection (liver). In another model, extravascular fluid levels, measured by bioassay of implanted paper discs, were compared with plasma levels in relation to control of a localized Staphylococcus aureus infection in rats. Extravascular fluid levels of azithromycin were greater than the MIC of the strain used for five days after a 100 mg/kg dose, while erythromycin levels were less than 20% of the MIC at 30 h after a 200 mg/kg dose. Serum concentrations of both compounds were less than 20% of the MIC at the time of challenge. The antibiotic levels at the site of infection correlated with the reduction of Staph. aureus cfu (99% with azithromycin compared with controls, P less than 0.01; 0% with erythromycin) recovered from inoculated discs. The significance of extravascular concentrations of azithromycin was further supported in other models of localized infections induced with Escherichia coli or a mixture of Staph. aureus and Bacteroides fragilis.

Effects of environmental factors on the in vitro potency of azithromycin.

The effects of media, pH, cations, serum, CO2 or anaerobic atmosphere, inoculum size and time of incubation on the in vitro potency of azithromycin were determined. The potency of azithromycin against all genera was particularly sensitive to changes in pH. The MIC for Staphylococcus aureus strains ranged from 50 micrograms/ml at pH 6 to less than or equal to 0.025 micrograms/ml at pH 8; for erythromycin the MIC change was less (1.6 to 0.05 micrograms/ml). Incubation for 18 h in 5% CO2 or an anaerobic atmosphere (10% CO2, 10% H2, 80% N2) lowered the pH by approximately 0.8 units with gram-negative organisms and 0.4 units with gram-positive organisms. This resulted in an MIC eight times greater than the aerobic MIC. In addition, the MIC100 for azithromycin and erythromycin against Bacteroides strains growing in Wilkins-Chalgren broth fell from 3.1 micrograms/ml in the anaerobic atmosphere to 0.2 and 0.4 micrograms/ml, respectively, when using the Oxyrase enzyme system to remove oxygen. With the Oxyrase system, the pH of the medium at the MIC remained at 7.2, while it fell to 6.7 in the anaerobic gas mixture. An increase in potency for both agents was also observed with other anaerobic species when using the Oxyrase system. The addition of serum produced an increase in potency of azithromycin and erythromycin that correlated with an increase in pH during incubation, despite the use of buffered media. Adding cations to Mueller-Hinton broth resulted in increased MICs for gram-negative organisms; the highest increases observed were four-fold for Escherichia coli.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of azithromycin, roxithromycin, and cephalexin penetration kinetics in early and mature abscesses.

During the process of abscess formation, a myriad of changes are observed histologically that impede the penetration of antimicrobial agents into infection loci. A Staphylococcus aureus foreign body abscess, developed in rats, was employed to evaluate the penetration kinetics of azithromycin, roxithromycin and cephalexin at various stages of abscess development; the progressive patho-histological changes of abscess formation were also characterized in this model. In an early abscess (18 h post-challenge), azithromycin penetration into inflammatory fluid was enhanced (AUC of 351 vs 130 mg.h/kg) and residence prolonged relative to an inflammation control (half-life of 88 vs 27 h). In contrast, roxithromycin and cephalexin penetration into, and residence in, inflammatory fluid were unaltered in the early abscess. However, penetration into, and egress from, a mature abscess (ten days post-challenge) were impeded for all three antimicrobials (P < or = 0.03). The penetration kinetics of azithromycin into inflammatory fluid in an early abscess were independent of the dose regimen, but dependent on the total dose. The persistently high concentrations of azithromycin in inflammatory fluid within abscess were associated with the infiltration of phagocytic cells and encapsulation by fibrous tissue. These data are consistent with a phagocytic delivery mechanism for azithromycin, whereby the presence of high concentrations of azithromycin in inflammatory fluid are a consequence of augmented drug distribution via the release of accumulated intracellular drug from the infiltrating phagocytic cells and fibroblasts associated with abscess formation.