George H. Miller

Antibiotic Susceptibility Profiles of Escherichia coli Strains Lacking Multidrug Efflux Pump Genes

ABSTRACT The contribution of seven known and nine predicted genes or operons associated with multidrug resistance to the susceptibility of Escherichia coli W3110 was assessed for 20 different classes of antimicrobial compounds that include antibiotics, antiseptics, detergents, and dyes. Strains were constructed with deletions for genes in the major facilitator superfamily, the resistance nodulation-cell division family, the small multidrug resistance family, the ATP-binding cassette family, and outer membrane factors. The agar dilution MICs of 35 compounds were determined for strains with deletions for multidrug resistance (MDR) pumps. Deletions in acrAB or tolC resulted in increased susceptibilities to the majority of compounds tested. The remaining MDR pump gene deletions resulted in increased susceptibilities to far fewer compounds. The results identify which MDR pumps contribute to intrinsic resistance under the conditions tested and supply practical information useful for designing sensitive assay strains for cell-based screening of antibacterial compounds.

Synthesis and spectrum of the neoglycoside ACHN-490.

ABSTRACT ACHN-490 is a neoglycoside, or “next-generation” aminoglycoside (AG), that has been identified as a potentially useful agent to combat drug-resistant bacteria emerging in hospitals and health care facilities around the world. A focused medicinal chemistry campaign produced a collection of over 400 sisomicin analogs from which ACHN-490 was selected. We tested ACHN-490 against two panels of Gram-negative and Gram-positive pathogens, many of which harbored AG resistance mechanisms. Unlike legacy AGs, ACHN-490 was active against strains expressing known AG-modifying enzymes, including the three most common such enzymes found in Enterobacteriaceae. ACHN-490 inhibited the growth of AG-resistant Enterobacteriaceae (MIC90, ≤4 μg/ml), with the exception of Proteus mirabilis and indole-positive Proteae (MIC90, 8 μg/ml and 16 μg/ml, respectively). ACHN-490 was more active alone in vitro against Pseudomonas aeruginosa and Acinetobacter baumannii isolates with AG-modifying enzymes than against those with altered permeability/efflux. The MIC90 of ACHN-490 against AG-resistant staphylococci was 2 μg/ml. Due to its promising in vitro and in vivo profiles, ACHN-490 has been advanced into clinical development as a new antibacterial agent.

In vitro and in vivo activities of SCH 56592 (Posaconazole), a new triazole antifungal agent, against Aspergillus and Candida

ABSTRACT SCH 56592 (posaconazole), a new triazole antifungal agent, was tested in vitro, and its activity was compared to that of itraconazole against 39 Aspergillus strains and to that of fluconazole against 275 Candida and 9 Cryptococcus strains. The SCH 56592 MICs for Aspergillus ranged from ≤0.002 to 0.5 μg/ml, and those of itraconazole ranged from ≤0.008 to 1 μg/ml. The SCH 56592 MICs for Candida andCryptococcus strains ranged from ≤0.004 to 16 μg/ml, and those of fluconazole ranged from ≤0.062 to >64 μg/ml. SCH 56592 showed excellent activity against Aspergillus fumigatus andAspergillus flavus in a pulmonary mouse infection model. When administered therapeutically, the 50% protective doses (PD50s) of SCH 56592 ranged from 3.6 to 29.9 mg/kg of body weight, while the PD50s of SCH 56592 administered prophylactically ranged from 0.9 to 9.0 mg/kg; itraconazole administered prophylactically was ineffective (PD50s, >75 mg/kg). SCH 56592 was also very efficacious against fluconazole-susceptible, -susceptible dose-dependent, or -resistantCandida albicans strains in immunocompetent or immunocompromised mouse models of systemic infection. The PD50s of SCH 56592 administered therapeutically ranged from 0.04 to 15.6 mg/kg, while the PD50s of SCH 56592 administered prophylactically ranged from 1.5 to 19.4 mg/kg. SCH 56592 has excellent potential for therapy against seriousAspergillus or Candida infections.

Pharmacokinetics of SCH 56592, a new azole broad-spectrum antifungal agent, in mice, rats, rabbits, dogs, and cynomolgus monkeys.

ABSTRACT SCH 56592 is a new broad-spectrum azole antifungal agent that is in phase 3 clinical trials for the treatment of serious systemic fungal infections. The pharmacokinetics of this drug candidate were evaluated following its intravenous (i.v.) or oral (p.o.) administration as a solution in hydroxypropyl-β-cyclodextrin (HPβCD) or oral administration as a suspension in 0.4% methylcellulose (MC) in studies involving mice, rats, rabbits, dogs, and cynomolgus monkeys. SCH 56592 was orally bioavailable in all species. The oral bioavailability was higher with the HPβCD solution (range, 52 to ∼100%) than from the MC suspension (range, 14 to 48%) and was higher in mice (∼100% [HPβCD] and 47% [MC]), rats (∼66% [HPβCD] and 48% [MC]), and dogs (72% [HPβCD] and 37% [MC]) than in monkeys (52% [HPβCD] and 14% [MC]). In rabbits, high concentrations in serum suggested good oral bioavailability with the MC suspension. The i.v. terminal-phase half-lives were 7 h in mice and rats, 15 h in dogs, and 23 h in monkeys. In rabbits, the oral half-life was 9 h. In species given increasing oral doses (mice, rats, and dogs), serum drug concentrations were dose related. Food produced a fourfold increase in serum drug concentrations in dogs. Multiple daily doses of 40 mg of SCH 56592/kg of body weight for eight consecutive days to fed dogs resulted in higher concentrations in serum, indicating accumulation upon multiple dosing, with an accumulation index of approximately 2.6. Concentrations above the MICs and minimum fungicidal concentrations for most organisms were observed at 24 h following a single oral dose in MC suspension in all five species studied (20 mg/kg for mice, rats, and rabbits and 10 mg/kg for dogs and monkeys), suggesting that once-daily administration of SCH 56592 in human subjects would be a therapeutically effective dosage regimen.

Correlation between aminoglycoside resistance profiles and DNA hybridization of clinical isolates.

DNA hybridization data and aminoglycoside resistance profiles (AGRPs) were determined for 4,088 clinical isolates from three studies (United States, Belgium, and Argentina). The correlation between susceptibility profiles and hybridization results was determined with nine DNA probes. For each of the seven aminoglycoside resistance profiles which we were able to test, the data suggested at least two distinct genes could encode enzymes which lead to identical resistance profiles. Furthermore, the DNA hybridization data showed that individual strains carried up to six unique aminoglycoside resistance genes. DNA hybridization revealed interesting differences in the frequencies of these genes by organism and by country.

Combating evolution with intelligent design: the neoglycoside ACHN-490.

The challenge posed by increasing levels of drug-resistant bacteria world-wide is manifest, and must be dealt with both by new approaches to the use of existing antibiotics and by the introduction of novel drugs. ACHN-490 is the first neoglycoside, or next-generation aminoglycoside, to begin clinical development. ACHN-490 was designed to target key pathogens, particularly gram-negative organisms and those resistant to older antibiotics. ACHN-490 demonstrates promising in vitro activity against wild-type and resistant bacteria while retaining the favorable bactericidal and synergistic properties of the aminoglycoside class. These attributes, along with the results of Phase 1 studies of ACHN-490 injection, suggest that ACHN-490 may help to fill the growing unmet need for new antibacterial agents.

KIJANIMICIN (Sch 25663), A NOVEL ANTIBIOTIC PRODUCED BY ACTINOMAD URA KIJANIATA SCC 1256

A novel antibiotic complex has been isolated form the fermentation broth of a new species of Actinomadura, A. kijaniata SCC 1256. The complex was separated form the broth by a solvent extraction procedure and consists of 1 major component, designated kijanimicin, and 3 minor components. Kijanimicin was isolated form the complex by column chromatography and/or preparative high pressure liquid chromatography. Structurally the compound is a unique, large acid enol antibiotic and possesses an unusual in vitro spectrum of activity against some Gram-positive and anaerobic microorganisms. In vivo it has also shown interesting activity against malaria.

Renal Extraction of Gentamicin in Anesthetized Dogs

The tubular handling of gentamicin (G) and its intrarenal distribution were determined to elucidate the mechanism of G accumulation in the kidney. At a serum level of 11.1 ± 0.5 μg/ml (10 animals), as maintained by constant infusion for 5 h, serum Na+ and K+, arterial pressure, effective renal plasma flow and glomerular filtration rate remained undisturbed. The clearance values in milliliters per minute for G, inulin, and p-aminohippuric acid were 40.3 ± 1.8, 49.9 ± 2.8, and 132 ± 14, respectively. The ratio of clearance of G to clearance of inulin was 0.82 ± 0.04 (P < 0.005), suggesting net reabsorption of G by the renal tubules. The renal cortex/serum ratio for G was 11.9 ± 2.1, and the medulla/serum ratio was 2.7 ± 0.4, indicating greater uptake of G by the cortex. The extraction ratio of p-aminohippuric acid was 0.74 ± 0.03. In contrast, the extraction ratio of G was 0.20 ± 0.03, which was significantly lower than that of inulin (0.30 ± 0.04). It is concluded that the accumulation of G in the cortex was due to tubular reabsorption. Probably some of the reabsorbed G became trapped in the epithelial cells after crossing the luminal membrane, whereas some returned to the circulation.

RENAL UPTAKE AND NEPHROTOXICITY OF GENTAMICIN DURING URINARY ALKALINIZATION IN RATS

1. Effect of urine pH on accumulation of gentamicin in the renal cortex of rats was studied following constant intravenous infusion, and single or repeated i.v. injections with gentamicin.

Renal Pharmacology of Netilmicin

Netilmicin (Sch 20569), a new semisynthetic aminoglycoside, was studied for its effects on kidney function and mechanisms by which it is handled by the kidneys. Measurements of glomerular filtration rate (GFR) and urinalysis in chronic rat studies indicated that the nephrotoxicity of netilmicin was remarkably less than that of gentamicin. Gentamicin caused a dose-related reduction in GFR in association with glucosuria and elevated fractional excretion of K+. By contrast, high doses of netilmicin produced only slight reduction in GFR with increased fractional excretion of K+ but without glucosuria. In separate experiments, rats were shown to excrete 71 to 90% of netilmicin or gentamicin in 24 h after daily intramuscular administration of doses of 20 or 40 mg/kg for 4 days. In acute experiments on anesthetized dogs, GFR and renal plasma flow were unaffected at serum levels of 11.0 ± 0.6 μg/ml maintained by constant infusion of netilmicin for 5 h. The renal clearance of netilmicin was significantly correlated with GFR. The urinary output of netilmicin was 80.0 ± 4.2% of the infusion rate and was independent of urine flow over the range of 0.04 to 0.33 ml/kg per min. Preferential accumulation of netilmicin occurred in the renal cortex; the cortex–serum and medulla–serum ratios were 9.9 ± 1.2 and 4.2 ± 0.6, respectively. In addition, the extraction ratio of netilmicin, which was lower than that of inulin, suggested that netilmicin reabsorption occurs in the proximal tubule and results in cortical accumulation. It is concluded that netilmicin, like gentamicin, is excreted by the dog kidney by glomerular filtration plus limited reabsorption. However, the new drug is characterized by low intrinsic nephrotoxicity in rats.