Siderophores as drug delivery agents: application of the "Trojan Horse" strategy.

The outer membrane permeability barrier is an important resistance factor of bacterial pathogens. In combination with drug inactivating enzymes, target alteration and efflux, it can increase resistance dramatically. A strategy to overcome this membrane-mediated resistance is the misuse of bacterial transport systems. Most promising are those for iron transport. They are vital for virulence and survival of bacteria in the infected host, where iron depletion is a defense mechanism against invading pathogens. We synthesized biomimetic siderophores as shuttle vectors for active transport of antibiotics through the bacterial membrane. Structure activity relationship studies resulted in siderophore aminopenicillin conjugates that were highly active against Gram-negative pathogens which play a crucial role in destructive lung infections in cystic fibrosis patients and in severe nosocomial infections. The mechanism of action and the uptake of the compounds via specific iron siderophore transport routes were demonstrated. The novel conjugates were active against systemic Pseudomonas aeruginosa infections in mice with ED(50) values comparable to the quinolone ofloxacin and show low toxicity.

Urinary bactericidal activity, urinary excretion and plasma concentrations of gatifloxacin (400 mg) versus ciprofloxacin (500 mg) in healthy volunteers after a single oral dose.

In an open randomised double-crossover study 12 volunteers (six men, six women) received a single oral dose of gatifloxacin (400 mg) or ciprofloxacin (500 mg) to assess urinary bactericidal activity (in eight intervals up to 120 h) and pharmacokinetic (PK) parameters (up to 36 h). Plasma concentrations and urinary excretion were determined by HPLC with fluorescence detection, and urinary bactericidal titers (UBT) by microdilution-method, using antibiotic-free urine of each volunteer. The mean maximum plasma concentration of gatifloxacin was 3.35 mg/l and that of ciprofloxacin 2.12 mg/l. The mean (median) cumulative renal excretion of the parent drug was for gatifloxacin 81 (83)% of the administered dose within 120 h and for ciprofloxacin 43 (45)%. The UBTs, i.e. the highest two-fold dilution (antibiotic-free urine as diluent) of urine still being bactericidal, were determined for an Escherichia coli ATCC reference strain and nine clinical uropathogens with the following MICs (mg/l) for gatifloxacin/ciprofloxacin (microdilution, MHB): E. coli ATCC 25922 (0.008/0.008); E. coli 523 (0.06/0.06); Klebsiella pneumoniae 1058 (0.03/0.016); Proteus mirabilis 524 (0.125/0.016); Pseudomonas aeruginosa 561 (1/0.125); Enterococcus faecalis strains 60 an 55 (0.5/1 and 8/32); Staphylococcus aureus strains 248 and 596 (both 0.03/0.125) and S. saprophyticus Ho94 (0.125/0.25). The median UBTs measured within the first 6h for gatifloxacin were between 1:16 and 1:>or=1024 for the Gram-negative strains including P. aeruginosa and between 1:8 and 1:>or=1024 for the five Gram-positive strains. The median UBTs for ciprofloxacin were between 1:64 and 1:>or=1024 for the Gram-negative strains (incl P. aeruginosa) and between 1:1.5 and 1:768 for the five Gram-positive strains. The UBTs up to 12 < 0.05 h showed no difference (P<0.05 ) for both E. coli strains, but ciprofloxacin was superior to gatifloxacin against Klebsiella, Proteus and Pseudomonas strains and gatifloxacin was superior to ciprofloxacin against all Gram-positive strains. For the UBTs at 12-24 h, gatifloxacin was generally superior to ciprofloxacin, but showed no difference in the Proteus and Pseudomonas strains. The areas under the UBT-time-curve (AUBT) up to 120 h showed statistically significant (P ) differences between both quinolones in favour of gatifloxacin against 8 of 10 strains tested, no difference for P. mirabilis and significantly higher activity of ciprofloxacin against P. aeruginosa. In conclusion, gatifloxacin and ciprofloxacin had overall comparable initial urinary bactericidal activity with some differences for specific pathogens, some times in favour of gatifloxacin (Gram-positives) and some times of ciprofloxacin (usually Gram-negatives), suggesting that for empiric therapy a single oral dose of gatifloxacin (400mg) would be clinically equivalent to ciprofloxacin (500 mg) twice daily-in agreement with the results of a clinical study in complicated UTI performed previously [Int. J. Antimicrob. Agents (2004)].

Catecholates and mixed catecholate hydroxamates as artificial siderophores for mycobacteria.

Different mono-, bis- or triscatecholates and mixed mono- or biscatecholate hydroxamates were synthesized as potential siderophores for mycobacteria. SiderOphore activity was tested by growth promotion assays using wild type strains and iron transport mutants of mycobacteria as well as Gram-negative bacteria. Some triscatecholates and biscatecholate hydroxamates were active in mutants of Mycobacterium smegmatis deficient in mycobactin and exochelin biosynthesis or exochelin permease, respectively, indicating an uptake route independent of the exochelin/mycobactin pathway. Structure activity relationships were studied. Ampicillin conjugates of some of these compounds were inactive against mycobacteria but active against Gram-negative bacteria.

Synthesis and biological activity of tris- and tetrakiscatecholate siderophores based on poly-aza alkanoic acids or alkylbenzoic acids and their conjugates with beta-lactam antibiotics.

New linear and tripodal tri-aza- and tetra-aza alkanoic acids or alkylbenzoic acids were prepared as basic structures for siderophore mimetics from polyamines and oxocarbonic acids or formylbenzoic acids by catalytic hydrogenation. From these acids acetylated tris- and tetrakiscatecholates or 8-acyloxy-2,4-dioxo-benzoxazine derivatives as well as compounds with spacer groups were synthesized. These derivatives were coupled with ampicillin, amoxicillin, bacampicillin or cefaclor to new siderophore antibiotic conjugates. Most of the catecholate derivatives showed high siderophore activities in strains of Pseudomonas aeruginosa and Escherichia coli in a growth promotion assay under iron limitation conditions. The beta-lactam conjugates were highly active in vitro against Gram-negative bacteria correlating to the siderophore activity of the catecholate moiety and depending on the beta-lactam part. One ampicillin conjugate based on 5-(aminoethyl)-2,5,8-triazaalkylbenzoic acid was highly active against Gram-negative and Gram-positive bacteria. It was shown that conjugates with enhanced activity against Gram-negative bacteria use active iron uptake routes to penetrate the bacterial outer membrane barrier. Correlations between structure and biological activity were studied.

Highly antibacterial active aminoacyl penicillin conjugates with acylated bis-catecholate siderophores based on secondary diamino acids and related compounds.

New acylated bis-catecholates and 1,3-benzoxazine-2,4-dione derivatives based on secondary diamino acids (N-(aminoalkyl)glycines, N-aminopropyl-alanine, and N-aminopropyl-4-aminovaleric acid), on N-(aminoalkyl)aminomethyl benzoic acids, on N-(aminoalkyl)aminomethyl phenoxyacetic acids, or on 3,5-diaminobenzoic acid were synthesized as artificial siderophores. The corresponding diamino acids were obtained from the diamines and oxocarboxylic acids by catalytic hydrogenation. The acylated bis-catecholates and 1,3-benzoxazine-2,4-diones were coupled with ampicillin or amoxicillin to new siderophore aminoacylpenicillin conjugates. These conjugates exhibited very strong antibacterial activity in vitro against Gram-negative bacterial pathogens including Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Escherichia coli, Klebsiella pneumoniae, and Serratia marcescens. The ampicillin derivative 7b (HKI 9924154) and the corresponding amoxicillin derivative 8 (HKI 9924155) represent the most active compounds. The conjugates can use bacterial iron siderophore uptake routes to penetrate the Gram-negative outer membrane permeability barrier. This was demonstrated by assays with mutants deficient in components of the iron transport systems. New siderophore penicillin V conjugates with the siderophore component attached to the phenyl ring of penicillin V are inactive against these Gram-negative bacteria.