Antibacterial agents that inhibit lipid A biosynthesis.

Lipid A constitutes the outer monolayer of the outer membrane of Gram-negative bacteria and is essential for bacterial growth. Synthetic antibacterials were identified that inhibit the second enzyme (a unique deacetylase) of lipid A biosynthesis. The inhibitors are chiral hydroxamic acids bearing certain hydrophobic aromatic moieties. They may bind to a metal in the active site of the deacetylase. The most potent analog (with an inhibition constant of about 50 nM) displayed a minimal inhibitory concentration of about 1 microgram per milliliter against Escherichia coli, caused three logs of bacterial killing in 4 hours, and cured mice infected with a lethal intraperitoneal dose of E. coli.

Reduced immunotoxicity and preservation of antibacterial activity in a releasable side-chain carbapenem antibiotic.

A carbapenem antibiotic, L-786,392, was designed so that the side chain that provides high-affinity binding to the penicillin-binding proteins responsible for bacterial resistance was also the structural basis for ameliorating immunopathology. Expulsion of the side chain upon opening of the beta-lactam ring retained antibacterial activity while safely expelling the immunodominant epitope. L-786,392 was well tolerated in animal safety studies and had significant in vitro and in vivo activities against methicillin- and vancomycin-resistant Staphylococci and vancomycin-resistant Enterococci.

(Acyloxy)alkyl carbamate prodrugs of norfloxacin.

As a new prodrug approach to norfloxacin (NFLX) we prepared the acetoxyalkyl carbamates of the type NFLX-CO-OCHR-OAc by the reaction of sodium or mercuric acetate on NFLX alpha-chloroalkyl carbamates. These produrgs did not have the bitter taste of NFLX. In vitro, the acetoxyethyl carbamate exhibited activity only against Staphylococcus spp. and was inactive against Gram-negative organisms. However, in the presence of serum and intestinal homogenate, esterase-catalyzed hydrolysis of the ester bond in these modified carbamates led to a cascade reaction resulting in the rapid regeneration of NFLX. At high oral doses of the prodrug, the acetaldehyde produced as a side product in the breakdown of the promoiety caused a slight decrease in alcohol metabolism in a mouse model. The bioavailability of NFLX from the acetoxyethyl carbamate was lower compared to an equivalent dose of NFLX when given as an oral suspension in rhesus monkeys, presumably because of the lower aqueous solubility of the prodrug.

The enhancement of beta-lactam antibiotic therapy by novobiocin.

Novobiocin demonstrates an effect similar to that of probenecid (the "probenecid effect") in enhancing the therapeutic efficacy of antibiotics excreted mainly by the renal tubules. The ability of cefoxitin, cephalexin, cephalothin and penicillin G to protect mice against infection with Salmonella schottmuelleri was enhanced 2- to 3-fold when the animals were given oral doses of either probenecid or of novobiocin. The efficacy of cephaloridine, excreted mainly by glomerular filtration, was not enhanced by either probenecid or by novobiocin.

Quaternary heterocyclylamino beta-lactams. IV. Comparison of the in vivo antibacterial activities of L-640,876, mecillinam, cefoxitin and cefotaxime.

The novel beta-lactam, L-640,876, exhibited excellent therapeutic activity when administered parenterally but not orally to mice infected with a variety of pathogenic bacteria. In this respect, the compound was as potent as cefotaxime against representative Gram-positive and Gram-negative organisms, in most cases, equal to or more potent than cefoxitin, and more effective than mecillinam. When administered subcutaneously to normal mice at dose levels ranging from 10 to 50 mg/kg, L-640,876 provided an adequate dose response, recovery of ca. 45% of biological activity in the urine, and excellent distribution at the highest dose level into liver, lung, kidney, heart muscle, but not brain.

Quaternary heterocyclylamino beta-lactams. VI. In vitro and in vivo antibacterial properties of L-642,946 and L-652,813, a long acting cephem.

Two newly described quaternary heterocyclylamino beta-lactams, L-642,946 and L-652,813, were shown to exhibit potent activity against a broad spectrum of aerobic and anaerobic bacteria in vitro. The activity of these agents in vitro translated well to chemotherapeutic activity in experimental bacteremias in mice. Substitution of the thiadiazine moiety of L-642,946 with a triazine moiety effected a marked change in the pharmacokinetics of the new derivative, L-652,813. In mice given a 20 mg/kg subcutaneous dose, the peak serum concentration and the half-life of L-652,813 were about three times greater than those of L-642,946 and the area under the serum concentration/time curve was increased by about 5-fold. The pharmacokinetics of L-652,813 in mice and in rhesus monkeys more closely resembled those of ceftriaxone which carries the same triazine moiety on the C-3 side chain.

Quaternary heterocyclylamino beta-lactams. III. The mode of action of L-640,876 and the effect of NaCl on membrane permeability and binding.

L-640,876, 7-beta(1-benzylpyridinium-4-yl)amino-3-[( (1-methyl-1 H-tetrazol-5-yl)thio]methyl)-ceph-3-em-4-carboxylate, is a potent representative of a new family of beta-lactam antibiotics which are similar in some respects to mecillinam. When L-640,876 and mecillinam were compared for effects on growth and morphology of Escherichia coli, it was observed that both drugs caused the formation of lemon-shaped cells during the first 30 minutes of exposure and during this period the culture turbidity increased without an appreciable change in culture viability. Unlike mecillinam, after 60 minutes of exposure to L-640,876 the majority of the lemon-shaped cells transformed into spindle-shaped cells and in the continuing presence of the drug formed osmotically fragile spheroplasts. Membrane binding studies indicated that, like mecillinam, L-640,876 was bound to the PBP-2 of E. coli and Proteus morganii; however, some binding of L-640,876 to the PBP-3 of E. coli was detected. In Staphylococcus aureus binding differences were more evident as L-640,876 was more rapidly bound to PBP-1 and 2 whereas mecillinam was rapidly bound to PBP-3. The reversal of inhibition of certain strains of Gram-negative bacteria by high ionic strength media could not be directly attributed to a reversal of antibiotic binding to the PBPs. Permeability studies indicated that the superior potency of L-640,876 in E. coli was partly due to its higher concentration in the periplasm which was unaffected by the simultaneous addition of drug and NaCl, however, in cells cultured in high ionic strength medium there was a marked reduction in penetration rate of all beta-lactams tested.

L-656,575 (OCP-9-176): a novel oxacephem. Pharmacokinetics and experimental chemotherapy.

L-656,575 is a new oxacephem that, based on studies in rhesus monkeys, is expected to have a moderately long half-life in humans. After administration of a 10-mg/kg dose by the intramuscular route to rhesus monkeys, peak serum concentrations of 32-54 micrograms/ml were seen at about 30 minutes, and the half-life was estimated to be 63 minutes. Urinary recovery of administered dose was greater than 94% in 6 hours. In mice given a 20-mg/kg dose by the subcutaneous route, a peak serum concentration of 22.9 microgram/ml was observed at 15 minutes after dosing, and the half-life in serum was about 18 minutes. Urinary recovery of the dose was 59% in 6 hours. In another study in mice, administration of probenecid did not extend the half-life of L-656,575, suggesting that the antibiotic is excreted primarily by glomerular filtration in this species. Binding to human plasma proteins was 30% at drug concentrations from 25-100 micrograms/ml. L-656,575 also was shown to be efficacious in experimental bacteremias due to Gram-positive and Gram-negative pathogens in mice, thus confirming the broad spectrum of activity demonstrated for L-656,575 in vitro.

L-658,310, a new injectable cephalosporin. II. In vitro and in vivo interactions between L-658,310 and various aminoglycosides or ciprofloxacin versus clinical isolates of Pseudomonas aeruginosa.

Combinations of L-658,310 and an aminoglycoside or ciprofloxacin were tested against clinical isolates of Pseudomonas aeruginosa using a checkerboard broth dilution technique. Using the mean fractional bactericidal concentration of less than or equal to 0.5 as the criterion for synergy, the combinations L-658,310/tobramycin and L-658,310/ciprofloxacin against strains of P. aeruginosa resistant to the companion drug were synergistic. The data plotted as isobolograms showed synergy for all combinations tested. Synergy was clearly demonstrated in time-kill experiments. A greater than 3-log decrease in viable cell count for P. aeruginosa was seen after exposure for 24 hours to subinhibitory concentrations of the combined agents. In in vivo mouse models, the efficacy of L-658,310 against experimental P. aeruginosa bacteremias was enhanced by the addition of a low dose of an aminoglycoside to the treatment regimen, thus confirming the synergy demonstrated in time-kill experiments.

Difficidin and oxydifficidin: novel broad spectrum antibacterial antibiotics produced by Bacillus subtilis. II. Isolation and physico-chemical characterization.

The isolation of difficidin (1) and oxydifficidin (2) from fermentation broth of Bacillus subtilis ATCC 39320 and the physico-chemical characterization of these labile antibiotics are described. The structures of the compounds represent a new class of antibiotics, characterized as highly unsaturated 22-membered macrolide phosphates. Difficidin and oxydifficidin undergo reversible thermal isomerization to 3 and 4 respectively. Biological evaluation of the isomers is presented.

L-658,310, a new injectable cephalosporin. III. Experimental chemotherapeutics and pharmacokinetics in laboratory animals.

The therapeutic activity of L-658,310 was demonstrated in experimental bacteremias in normal, diabetic and neutropenic mice. Especially potent activity was shown against the usually difficult to control pathogens, Enterobacter cloacae and Pseudomonas aeruginosa, that were resistant to ceftazidime and/or gentamicin. Pharmacokinetic studies in mice showed a linear dose response in serum after the 20 and 50 mg/kg subcutaneous dose and urinary recoveries of administered dose of about 60% in 6 hours. Excretion was mainly by glomerular filtration. In a crossover design in rhesus monkeys, the pharmacokinetics of L-658,310 were similar to those of ceftazidime and suggest a moderately long half-life in serum of humans.

Difficidin and oxydifficidin: novel broad spectrum antibacterial antibiotics produced by Bacillus subtilis. I. Production, taxonomy and antibacterial activity.

Difficidin and oxydifficidin, two novel macrocyclic polyene lactone phosphate esters were discovered in fermentation broths of each of two strains of Bacillus subtilis: ATCC 39320 and ATCC 39374. Difficidin and oxydifficidin each showed a broad spectrum of activity against aerobic and anaerobic bacteria. Many of the susceptible aerobes and anaerobes were human pathogens resistant to one or more antibiotics. Difficidin and oxydifficidin when administered intraperitoneally protected mice against an otherwise lethal bacteremia caused by Klebsiella pneumoniae (ED50 in mg/kg of 1.31 and 15.6 respectively). Neither difficidin nor oxydifficidin were effective when administered via the subcutaneous route.

Comparative in-vitro activities of ertapenem against aerobic bacterial pathogens isolated from patients with complicated intra-abdominal infections.

The in vitro activities of ertapenem, ceftriaxone, amoxicillin-clavulanate, ampicillin-sulbactam, and piperacillin-tazobactam were compared against 1018 aerobic bacterial pathogens isolated from 531 patients with complicated intra-abdominal infection. Enterobacteriaceae accounted for 66.3% of the aerobic bacteria; Escherichia coli was the most common isolate. The ertapenem minimal inhibitory concentration was < or = 2 microg/mL for 74.6% of isolates and > or = 8 microg/mL for 21.9% (including isolates of enterococci, methicillin-resistant Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa). Against Enterobacteriaceae, ertapenem was the most potent and the most active drug evaluated (100% susceptible), followed by ceftriaxone (98% susceptible), piperacillin-tazobactam (96% susceptible), amoxicillin-clavulanate (80% susceptible), and ampicillin-sulbactam (64% susceptible). Piperacillin-tazobactam was the only drug evaluated with clinically useful activity against P. aeruginosa. In summary, ertapenem was highly active in vitro against many clinically important aerobic intra-abdominal bacterial pathogens, especially Enterobacteriaceae.

Efficacy of caspofungin against Aspergillus flavus, Aspergillus terreus, and Aspergillus nidulans.

The echinocandin caspofungin is a potent inhibitor of the activity of 1,3-beta-D-glucan synthase from Aspergillus flavus, Aspergillus terreus, and Aspergillus nidulans. In murine models of disseminated infection, caspofungin prolonged survival and reduced the kidney fungal burden. Caspofungin was at least as effective as amphotericin B against these filamentous fungi in vivo.

Cephamycins, a new family of beta-lactam antibiotics. 3. In vitro studies.

Cephamycins A, B, and C are naturally produced cephalosporin-type antibiotics. Although A and B were found to be more active than C against gram-positive organisms, they were not so active against such strains as are cephalosporin C or the semisynthetic antibiotics cephaloridine and cephalothin. Against gram-negative organisms, cephamycin C was more active than A or B and, in general, was as active as the cephalosporins. In addition, cephamycin C was active in vitro against clinically isolated strains resistant to the cephalosporins, such as Proteus, Providencia, and Escherichia coli. The in vitro antibacterial activity of cephamycin C, cephalothin, and cephaloridine is primarily bactericidal. A 10,000-fold increase in inoculum of a strain of Proteus mirabilis resulted in 200-fold or greater increases in minimal inhibitory and minimal bactericidal end points of cephalothin and cephaloridine, but only 10- and 16-fold increases, respectively, for cephamycin C. After 15 passages through antibiotic-containing broths, during which time a culture of E. coli showed an increase in minimal inhibitory concentrations of streptomycin of >1,000-fold, end points for cephamycin C increased 4-fold, for cephalothin, 1.5-to 6-fold, and for cephaloridine, 128-fold.

Correlation of in vitro susceptibility with in vivo efficacy in mice for cefoxitin in comparison with cephalosporins.

Agar minimal inhibitory concentrations and mouse protection test effective doses were determined for each of four beta-lactam antibiotics against each of 12 Gram-negative and 3 Gram-positive bacterial cultures. The beta-lactamase activity of these cultures also was studied. The data were examined to determine whether relative in vivo efficacies could be predicted from relative in vitro activities. Although such predictions were quite accurate for cefoxitin and cefazolin, this was not true for cefamandole or for cephalothin. Such poor predictability was not necessarily associated with the susceptibility of these cephalosporins to hydrolysis by bacterial beta-lactamases. Although the clinical significance of these observations is not known, these data emphasize that relative in vitro activities should be used only with caution to estimate in vivo efficacies, since not all compounds show the excellent predictability observed here for cefazolin and cefoxitin.

Preliminary animal pharmacokinetics of the parenteral antifungal agent MK-0991 (L-743,872).

MK-0991 (L-743,872) is a potent antifungal agent featuring long half-life pharmacokinetics. The pharmacokinetics of MK-0991 administered intravenously to mice, rats, rhesus monkeys, and chimpanzees is presented. Unique to MK-0991 is its consistent cross-species performance. The range of values for the pharmacokinetic parameters were as follows: clearance, 0.26 to 0.51 ml/min/kg; half-life, 5.2 to 7.6 h; and distributive volume, 0.11 to 0.27 liters/kg. The level of protein binding of MK-0991 was determined to be 96% in mouse and human serum. The compound exhibited high affinities for human serum albumin and at least two lipid components. The rationale for the selection of MK-0991 as a drug development candidate was based on its two- to threefold superior pharmacokinetic performance in chimpanzees over the performance of an otherwise equivalent analog, L-733,560. Once-daily dosing for MK-0991 is indicated by a graphical comparison of levels in the circulations of chimpanzees and mice. In a study of the pharmacokinetics of MK-0991 in mouse tissue, the organs were assayed following intraperitoneal administration. The area under the concentration-versus-time curves (AUC) segregated the tissues into three exposure categories relative to plasma. The tissues with greater exposure than that for plasma were liver (16 times), kidney (3 times), and large intestine (2 times). The exposure for small intestine, lung, and spleen were equivalent to that for plasma. Organs with lower levels of exposure were the heart (0.3 times that for plasma), thigh (0.2 times), and brain (0.06 times). Kinetically, drug was cleared more slowly from all tissues than from plasma, indicating that terminal-phase equilibrium had not been achieved by 24 h. Thus, some measure of accumulation is predicted for all tissues. Single daily doses of MK-0991 should provide adequate systemic levels of fungicidal activity as a result of its long half-life pharmacokinetics, wide distribution, and slowly accumulating concentrations in tissue.

Cephamycins, a new family of beta-lactam antibiotics. IV. In vivo studies.

Cephamycin A was found to be more active in vivo than cephamycin B. In comparison with cephamycin C, cephamycin A was more active against gram-positive organisms but less active against gram-negative organisms. Given subcutaneously, cephamycin C had good in vivo gram-negative activity, comparing favorably with cephalothin and cephaloridine against cephalosporin-susceptible organisms. In general, against the gram-negative organisms, it was more active than cephalothin or cephalosporin C and about as active as cephaloridine. In addition, cephamycin C protected mice against beta-lactamase-producing Proteus cultures, including clinically isolated strains. The compound is remarkably nontoxic. Cephamycin C was detected in the serum and recovered from the urine of treated mice to about the same extent as cephaloridine. Like cephaloridine and cephalosporin C, cephamycin C must be excreted mainly by glomerular filtration, because the use of probenecid did not enhance the therapeutic effectiveness nor concentrations of these agents in the sera of treated mice.

Cefoxitin, a semisynthetic cephamycin antibiotic: in vivo evaluation.

Cefoxitin, 3-carbamoyloxymethyl-7-alpha-methoxy-7-[2-(2-thienyl)acetamido]-3-cephem-4- carboxylic acid, a semisynthetic cephamycin antibiotic shown to have broad-spectrum activity in vitro, is active also in vivo against a wide variety of bacteria including penicillin-resistant staphylococci. It is, however, particularly effective against gram-negative organisms including strains of indole-positive Proteus against which cephalothin and cephaloridine are ineffective. When cefoxitin is given subcutaneously, concentrations in mouse blood, urine, and other tissues are higher than those seen for cephalothin. Higher concentrations in the blood and greater therapeutic efficacy are achieved with cefoxitin when it is given with probenecid. For this reason it is believed that cefoxitin is excreted mainly by way of the renal tubules. The data indicate that cefoxitin has potential as a therapeutically useful antibiotic.

Norfloxacin versus trimethoprim-sulphamethoxazole: efficacy in a model of ascending urinary tract infection in normal and streptozotocin-induced diabetic mice.

A model of ascending urinary tract infection due to an isolate of Escherichia coli was developed in normal and streptozotocin-induced mice to compare the efficacy of norfloxacin and trimethoprim-sulphamethoxazole. Norfloxacin and trimethoprim-sulphamethoxazole both were effective in reducing the number of colony forming units of E. coli from the kidneys of normal experimentally-infected mice, although norfloxacin yielded a greater quantitative reduction of colony forming units. Norfloxacin was substantially more effective than trimethoprim-sulphamethoxazole in reducing the number of colony forming units from kidney homogenates when the test animals were diabetic. This study supports the initiation of clinical trials to evaluate norfloxacin in diabetic patients.