Prabhavathi B. Fernandes

Comparative in vitro activities of new 14-, 15-, and 16-membered macrolides.

The in vitro activities of several 14-, 15- and 16-membered macrolides were compared with that of erythromycin. In general, 14-membered macrolides such as erythromycin, clarithromycin, and flurithromycin were more active against streptococci and Bordetella pertussis than was the 15-membered macrolide azithromycin, which was more active than 16-membered macrolides such as miocamycin and rokitamycin. Clarithromycin was the most active compound against Streptococcus pyogenes, pneumococci, Listeria monocytogenes, and Corynebacterium species. Legionella pneumophila was most susceptible to miocamycin, clarithromycin, and rokitamycin. Branhamella catarrhalis, Neisseria gonorrhoeae, and Haemophilus influenzae were most susceptible to azithromycin. Azithromycin and dirithromycin were the most active compounds against Campylobacter jejuni. MICs of 16-membered macrolides for strains expressing inducible-type resistance to erythromycin were less than or equal to 1 microgram/ml, whereas none of the compounds had activity against strains expressing constitutive-type resistance. The MICs of roxithromycin, miocamycin, rokitamycin, and josamycin increased in the presence of human serum, whereas MICs of the other compounds either were unchanged or decreased.

In vitro and in vivo activities of clarithromycin against Mycobacterium avium.

There is no effective therapy to treat Mycobacterium avium complex infection in patients with acquired immune deficiency syndrome. Clarithromycin (A-56268; TE-031) is a new macrolide which is twofold more active than erythromycin against most aerobic bacteria. In addition, higher levels in serum and tissue are achieved with clarithromycin than with erythromycin. In this study, clarithromycin, erythromycin, difloxacin, temafloxacin, ciprofloxacin, rifampin, amikacin, and ethambutol were tested in vitro and in vivo against the M. avium complex. The MICs for 90% of strains tested were 4 micrograms/ml for clarithromycin, 64 micrograms/ml for erythromycin, 32 micrograms/ml for difloxacin, 8 micrograms/ml for temafloxacin, 4 micrograms/ml for ciprofloxacin, 4 micrograms/ml for rifampin, 32 micrograms/ml for amikacin, and 32 micrograms/ml for ethambutol. Beige mice were infected intravenously with 10(7) CFU of M. avium ATCC 25291. Treatment was started on day 6 after infection and was administered twice a day at 8-h intervals for 9 days. Clarithromycin was the most effective compound in these tests and was effective in reducing the viable bacterial counts in the spleen when it was administered subcutaneously or orally at a dose of 25 mg/kg. Amikacin was the only other compound which showed activity in vivo. The peak concentration in serum at which clarithromycin was active was approximately 1.0 microgram/ml.

In vitro and in vivo evaluation of A-56268 (TE-031), a new macrolide.

The in vitro and in vivo antibacterial activity of A-56268 (TE-031), the 6-O-methyl derivative of erythromycin, was compared with those of erythromycin and other reference drugs. A-56268 had the same spectrum of antibacterial activity as erythromycin. A-56268 was generally 1 log2 dilution more potent or equal to erythromycin against all organisms except haemophilus influenzae and Propionibacterium acnes, for which A-56268 was 1 log2 dilution and 3 log2 dilutions, respectively, less potent. The MBC of A-56268 and erythromycin was not significantly different from the MIC against Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus epidermidis, and H. influenzae but was more than 2 log2 dilutions higher than the MICs for some Staphylococcus aureus strains. Human serum at a concentration of 50% did not change the in vitro potency of A-56268 or erythromycin. A-56268 was similar to erythromycin in being more active at pH 8.0 than at the physiologic pH of 7.3. The activity of A-56268 was synergistic with sulfamethoxazole against 4 of 12 strains of H. influenzae. In mouse protection tests, when administered orally A-56268 was more potent than erythromycin against H. influenzae, S. pyogenes, S. pneumoniae, and S. aureus. After subcutaneous administration the potencies of A-56268 and erythromycin were not statistically different from each other. A-56268 was more potent than erythromycin against Legionella infection in guinea pigs. The concentration of A-56268 in the serum and lung was higher than that of erythromycin after intraperitoneal administration. In A-56268 in the serum and lung was higher than that of erythromycin after intraperitoneal administration. In mice, the peak levels in serum of A-56268 and erythromycin were similar after subcutaneous administration and seven times higher for A-56268 after oral administration. The serum half-life of A-56268 was approximately twice that of erythromycin after administration by both routes.

In vitro and in vivo evaluation of tiacumicins B and C against Clostridium difficile.

Tiacumicins B and C are members of a novel group of 18-membered macrolide antibiotics with in vitro activity against Clostridium difficile. The MICs against 15 strains of C. difficile were 0.12 to 0.25 microgram/ml for tiacumicin B, 0.25 to 1 microgram/ml for tiacumicin C, and 0.5 to 1 microgram/ml for vancomycin. The resistance frequency for both compounds against C. difficile was less than 2.8 x 10(-8) at four and eight times the MIC. The in vivo activities of the tiacumicins against two strains of C. difficile were compared with that of vancomycin in a hamster model of antibiotic-associated colitis. Oral therapy with 0.2, 1, or 5 mg of tiacumicin B or C per kg of body weight protected 100% of clindamycin-treated hamsters exposed to C. difficile ATCC 9689. Oral treatment with identical doses of vancomycin produced a prolonged, dose-dependent survival of hamsters, but it did not prevent the development of fatal colitis at doses of up to 5 mg/kg. When clindamycin-treated animals were exposed to another strain of C. difficile, both tiacumicin B and vancomycin were protective at 5 mg/kg, but not at lower doses. Tiacumicin C was not tested in vivo against the second strain of C. difficile. No tiacumicin B or C was detected in the sera of hamsters treated with single oral doses of 25 mg/kg, while antibiotic levels in the ceca of these hamsters reached 248 micrograms/ml and 285 mg/ml for tiacumicins B and C, respectively. The tiacumicins demonstrated in vitro and in vivo potencies against C. difficile and achieved high concentrations in the cecum, but not the serum, of hamsters after oral administration.

Comparative antibacterial activities of temafloxacin hydrochloride (A-62254) and two reference fluoroquinolones.

The in vitro and in vivo properties of a new 1-difluorophenyl-6-fluoroquinolone, temafloxacin hydrochloride (A-62254), were compared with those of difloxacin and ciprofloxacin. Temafloxacin hydrochloride was as active as ciprofloxacin and difloxacin against staphylococci and as active as ciprofloxacin and 2 twofold dilutions more active than difloxacin against streptococci. Against gram-negative enteric bacteria and Pseudomonas aeruginosa, temafloxacin hydrochloride was 2 twofold dilutions more active than difloxacin but 2 to 4 twofold dilutions less active than ciprofloxacin. The MICs of temafloxacin hydrochloride and difloxacin were increased by 2 to 5 twofold dilutions in urine at pH 6.5 compared with 4 to 5 twofold-dilution increases in the MICs of ciprofloxacin. The MICs of temafloxacin hydrochloride, difloxacin, and ciprofloxacin were increased by 1 to 3 twofold dilutions in serum. The MICs of temafloxacin hydrochloride, difloxacin, and ciprofloxacin were the same or within 1 to 2 twofold dilutions at pHs 6.5, 7.2, and 8.0. When administered orally in mouse protection tests, temafloxacin hydrochloride was as active as difloxacin and 5 to 10 times more active than ciprofloxacin against infections with Staphylococcus aureus and streptococci. Against infections with gram-negative enteric bacteria and P. aeruginosa, temafloxacin hydrochloride was as active as difloxacin and ciprofloxacin. Temafloxacin hydrochloride was three times less active than difloxacin but was five times more active than ciprofloxacin against infections with Salmonella typhimurium. Temafloxacin hydrochloride was as active as difloxacin and ciprofloxacin against P. aeruginosa and Proteus mirabilis pyelonephritis in mice. The peak serum concentration and serum half-life of temafloxacin hydrochloride in mice were approximately one-half and one-sixth, respectively, that of difloxacin after oral administration. The peak serum concentration of temafloxacin hydrochloride in mice after oral administration was six times higher than that of ciprofloxacin, and the serum half-life was equal to that of ciprofloxacin.

In vitro evaluation of A-56619 (difloxacin) and A-56620: new aryl-fluoroquinolones.

The in vitro antibacterial potencies of A-56619 and A-56620, two new aryl-fluoroquinolones, were compared with the potency of norfloxacin against a broad spectrum of organisms. Cefotaxime, aztreonam, piperacillin, imipenem, penicillin, and gentamicin were also tested for reference purposes. The MICs required to inhibit at least 90% of the strains tested ranged from 0.25 to 4 micrograms/ml for A-56619 and from 0.06 to 0.5 microgram/ml for A-56620 for members of the Enterobacteriaceae. A-56619 was generally twofold less potent and A-56620 was twofold more potent than norfloxacin against most aerobic gram-negative bacilli, including members of the Enterobacteriaceae and Pseudomonas aeruginosa. Against indole-positive Proteus, Morganella, Providencia rettgeri, and Serratia strains, A-56619 was at least 8- to 16-fold less potent than norfloxacin. A-56619 and A-56620 were four- to eightfold more potent than norfloxacin against Staphylococcus aureus and equally potent to fourfold more potent against Streptococcus species, Haemophilus influenzae, and Neisseria gonorrhoeae. The MICs of A-56619 and A-56620 were only slightly affected by increased inoculum size or by the addition of various cations at physiologic concentrations. A-56619 was three- to fivefold less active at pH 8.0 than at pH 6.5 or 7.2. A-56620 was twofold less active at pH 6.5 than at pH 8.0 or 7.2 against members of the Enterobacteriaceae and Pseudomonas aeruginosa; similar pH variations did not affect A-56620 activity against gram-positive cocci. The potencies of A-56619, A-56620, and norfloxacin were less in urine than in Mueller-Hinton broth; however, this effect was more pronounced with norfloxacin. Human serum at a concentration of 50% caused a 4- to 64- fold decrease in the potency of A-56619 and an average 4-fold decrease in the potency of A-56620, compared with no effect on the potency of norfloxacin. A-56619, A-56620, and norfloxacin were bactericidal and, at four times the MIC, reduced the viable cell counts of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa by approximately 99.9% within 2 h. A-56619, A-56620, and norfloxacin showed no significant synergistic activity and no antagonism when they were aminoglycoside or beta-lactam antimicrobial agents.

Susceptibility testing of macrolide antibiotics against Haemophilus influenzae and correlation of in vitro results with in vivo efficacy in a mouse septicemia model.

There is poor correlation between the MICs and zone sizes obtained for erythromycin against Haemophilus influenzae. The effect of two media, Mueller-Hinton medium supplemented with 3% lysed horse blood and 10 micrograms of NAD per ml (MHA + LYHB) and Mueller-Hinton agar supplemented with 1% bovine hemoglobin and 1% IsoVitaleX (MHA + HGB), on the MICs and zone sizes of erythromycin against H. influenzae was determined. The effect of three different methods for inoculum preparation on the susceptibility of H. influenzae was also determined. The MICs were independent of the method of inoculum preparation, but the zone sizes were smaller if the inoculum was carefully adjusted to contain approximately 10(8) CFU/ml. MICs were higher and zone sizes were smaller when MHA + HGB was used instead of MHA + LYHB. Good correlation was found when MHA + LYHB was used for determining the MIC and MHA + HGB was used for determining susceptibility by the disk method. When the inoculum was adjusted to match a McFarland 0.5 standard, the viable counts had to be approximately 10(8) CFU/ml for good correlation between MICs and zone sizes. A-56268, a new macrolide antibiotic, was tested against H. influenzae, and its MICs and tentative breakpoints against this organism were determined. The MICs obtained by various methods were correlated with in vivo efficacy by using a mouse septicemia model. MICs obtained on MHA + HGB or MHA + LYHB incubated without a 5% CO2 atmosphere showed the best correlation with in vivo efficacy.

A-61827 (A-60969), a new fluoronaphthyridine with activity against both aerobic and anaerobic bacteria

A-61827 (A-60969 is the hydrochloric salt of A-61827) is a new aryl-fluoronaphthyridine which is active against aerobic and anaerobic bacteria. The MICs of A-61827 for 90% of strains (MIC90) of staphylococci and streptococci were less than or equal to 1 microgram/ml and were generally 1 to 4 twofold dilutions less than those of ciprofloxacin for these bacteria. The MIC90S of A-61827 for members of the family Enterobacteriaceae and Pseudomonas aeruginosa were also less than or equal to 1 microgram/ml. Ciprofloxacin was 1 to 3 twofold dilutions more active than A-61827 against these gram-negative bacteria. Neisseria gonorrhoeae, Campylobacter jejuni, and Haemophilus influenzae were susceptible to less than 0.06 microgram of A-61827 per ml. The MIC90 of A-61827 for Legionella pneumophila was 0.25 microgram/ml. A-61827 was as potent or 1 to 2 twofold dilutions more potent than ciprofloxacin against these organisms. The MIC90 of A-61827 for all anaerobic bacteria was less than or equal to 4 micrograms/ml compared with less than or equal to 32 micrograms/ml for ciprofloxacin. In mouse protection tests, A-61827 was as active as ciprofloxacin against Escherichia coli, P. aeruginosa, and Salmonella typhimurium and 5 to 10 times more active than ciprofloxacin against Staphylococcus aureus and Streptococcus pyogenes. A-61827 was as active as ciprofloxacin against P. aeruginosa in a mouse pyelonephritis model and more active than ciprofloxacin and metronidazole in a mouse Bacteroides fragilis abscess model. After oral administration of 100 mg/kg to mice, the peak concentrations of A-61827 and ciprofloxacin in serum were 2.3 and 2.4 micrograms/ml and the half-lives in serum were 3.9 and 1.2 h, respectively.

Microbial resistance: novel screens for a contemporary problem.

Historically, natural products have been the source of a large variety of antibacterial agents. In the 1980s, no additional useful antibacterial agents were discovered, leading to the belief that most useful chemotypes from natural product sources had already been discovered. At this time, advances in biotechnology made it feasible to produce sufficient enzyme to set up cell‐free screens. Chemical compound libraries and combinatorial synthesis became the source of chemical diversity for the screens. In spite of these efforts, very few new antibacterial agents have been discovered in the last decade. At Small Molecule Therapeutics, Inc., we have developed phenotype‐based screens that take advantage of the natural physiology and biochemistry of the target enzymes. We have developed a screen to identify bacterial DNA gyrase and topoisomerase IV poisons. The “hits” identified in this screen are being characterized further. A second screen has also been developed against bacterial topoisomerase 1 in which compounds that cause DNA damage through their interaction with bacterial topoisomerase 1 have been identified. Three of the compounds identified in the screen inhibit DNA relaxation mediated by bacterial topoisomerase 1, induce DNA cleavage, are noncytotoxic at >10μM, and have MICs of 4.0 μg/mL against Staphylococcus aureus.

Nature nurtures the design of new semi-synthetic macrolide antibiotics

Erythromycin and its analogs are used to treat respiratory tract and other infections. The broad use of these antibiotics during the last 5 decades has led to resistance that can range from 20% to over 70% in certain parts of the world. Efforts to find macrolides that were active against macrolide-resistant strains led to the development of erythromycin analogs with alkyl-aryl side chains that mimicked the sugar side chain of 16-membered macrolides, such as tylosin. Further modifications were made to improve the potency of these molecules by removal of the cladinose sugar to obtain a smaller molecule, a modification that was learned from an older macrolide, pikromycin. A keto group was introduced after removal of the cladinose sugar to make the new ketolide subclass. Only one ketolide, telithromycin, received marketing authorization but because of severe adverse events, it is no longer widely used. Failure to identify the structure-relationship responsible for this clinical toxicity led to discontinuation of many ketolides that were in development. One that did complete clinical development, cethromycin, did not meet clinical efficacy criteria and therefore did not receive marketing approval. Work on developing new macrolides was re-initiated after showing that inhibition of nicotinic acetylcholine receptors by the imidazolyl-pyridine moiety on the side chain of telithromycin was likely responsible for the severe adverse events. Solithromycin is a fourth-generation macrolide that has a fluorine at the 2-position, and an alkyl-aryl side chain that is different from telithromycin. Solithromycin interacts at three sites on the bacterial ribosome, has activity against strains resistant to older macrolides (including telithromycin), and is mostly bactericidal. Pharmaceutical scientists involved in the development of macrolide antibiotics have learned from the teachings of Professor Satoshi Omura and progress in this field was not possible without his endeavors.