Antibacterial efficacy against an in vivo Salmonella typhimurium infection model and pharmacokinetics of a liposomal ciprofloxacin formulation

Abstract

The fluoroquinolone antibiotic ciprofloxacin has been encapsulated into large unilamellar vesicles (LUV) at efficiencies approaching 100%. Drug accumulation proceeded in response to a transmembrane gradient of methylammonium sulfate and occurred concomitantly with the efflux of methylamine. A mechanism for the encapsulation process is described. LUV composed of dipalmitoylphosphatidylcholine-cholesterol (DPPC/chol), distearoylphosphatidylcholine-cholesterol (DSPC/chol), or sphingomyelin-cholesterol (SM/chol) increased the circulation lifetime of ciprofloxacin after intravenous (i.v.) administration by > 15-fold. The retention of ciprofloxacin in liposomes in the circulation decreased in the sequence SM/chol > DSPC/chol > DPPC/chol. Increased circulation lifetimes were associated with enhanced delivery of the drug to the livers, spleens, kidneys, and lungs of mice. Encapsulation of ciprofloxacin also conferred significant increases in the longevity of the drug in the plasma after intraperitoneal administration and in the lungs after intratracheal administration in comparison to free ciprofloxacin. The efficacy of a single i.v. administration of an SM/chol formulation of ciprofloxacin was measured in a Salmonella typhimurium infection model. At 20 mg of ciprofloxacin per kg of body weight, the encapsulated formulation resulted in 10(3)- to 10(4)-fold fewer viable bacteria in the livers and spleens of infected mice than was observed for animals treated with free ciprofloxacin. These results show the utility of liposomal encapsulation of ciprofloxacin in improving the pharmacokinetics, biodistribution, and antibacterial efficacy of the antibiotic. In addition, these formulations are well suited for i.v., intraperitoneal, and intratracheal or aerosol administration.

FIG. 1

Solubility of ciprofloxacin in aqueous solutions buffered in the pH range between 4 and 11. Ciprofloxacin hydrochloride was dissolved to saturation in 50 mM HEPES at various pHs. Excess drug was removed by centrifugation, and the supernatant was assayed for ciprofloxacin content by A₂₇₅.

FIG. 2

Loading of ciprofloxacin into liposomes. (A) Uptake of ciprofloxacin, expressed as the ciprofloxacin/lipid ratio (•) and release of [¹⁴C]methylamine, expressed as the methylamine/lipid ratio (○). Ciprofloxacin uptake was initiated by the addition of ciprofloxacin to 100-nm unilamellar liposomes composed of DPPC/chol (55/45 [mol/mol]) and possessing a transmembrane 0.3 M methylammonium sulfate gradient. (B) Calculated stoichiometry of moles of methylamine released/moles of ciprofloxacin loaded.

FIG. 3

Pharmacokinetics of free and liposomal ciprofloxacin after i.v. administration. (A) Concentrations of ciprofloxacin in plasma after i.v. administration of free ciprofloxacin (•) or ciprofloxacin encapsulated in liposomes comprised of DPPC/chol (▪), DSPC/chol (▴), or SM/chol (⧫). The dose of ciprofloxacin injected for all treatments was 15 mg/kg. (B) Ciprofloxacin/lipid ratios in plasma after i.v. administration of the DPPC/chol (▪), DSPC/chol (▴), or SM/chol (⧫) formulation of liposomal ciprofloxacin. Data represent means ± standard errors from three mice.

FIG. 4

Summary of the biodistribution of free and liposomal ciprofloxacin (cipro) after i.v. administration. Area under the concentration-time curve (AUC) values over 24 h for the different liposomal formulations are expressed relative to that for free ciprofloxacin in liver, lung, spleen, kidney, and muscle (A). Ciprofloxacin concentrations in the lungs of mice after i.v. administration of free ciprofloxacin (•) or ciprofloxacin encapsulated in SM/chol liposomes (⧫) are shown as an example (B). The dose of ciprofloxacin injected for all treatments was 15 mg/kg. Data represent means ± standard errors from three mice.

FIG. 5

Comparison of ciprofloxacin pharmacokinetics after i.v. and i.p. administration. (A) Levels of free (•,▪) or liposomal (▴,⧫) ciprofloxacin in plasma after i.v. (•,▴) or i.p. (▪,⧫) administration. Liposomes were composed of SM/chol, and the dose of ciprofloxacin injected for all groups was 15 mg/kg. (B) Ciprofloxacin/lipid ratios in plasma after i.v. (▴) or i.p. (⧫) administration of ciprofloxacin encapsulated in SM/chol liposomes. Data represent means ± standard errors from three mice.

FIG. 6

Pharmacokinetics after intratracheal administration. Quantities of ciprofloxacin (•,▪) and lipid (□) in the lungs of ICR mice after the intratracheal administration of either free ciprofloxacin (•) or ciprofloxacin encapsulated in liposomes comprised of DPPC/chol (▪,□) are shown. Data represent means ± standard errors from three mice.

FIG. 7

In vivo antibacterial efficacy against Salmonella typhimurium. The CFU of viable bacteria per milliliter of liver (open bars) or spleen (shaded bars) homogenates for animals infected with 66 to 88 CFU of Salmonella typhimurium were either left untreated, were treated once with saline, or were treated once with free or liposomal ciprofloxacin at 20 mg/kg. Data represent means ± standard errors from three mice.

FIG. 8

Schematic representation of the active loading method for ciprofloxacin. Ciprofloxacin exists in cationic, anionic, zwitterionic, and neutral forms. (Note that this ionization scheme is identical both inside and outside the liposome, but for clarity, is shown only on the outside.) Inside the liposome is a low internal pH as a consequence of methylammonium ionization to methylamine and a proton. The neutral form of ciprofloxacin is membrane permeable and crosses to the aqueous lumen of the liposomes, where the low pH favors the protonated species. Cationic ciprofloxacin cannot diffuse back out of the vesicle; consequently, the drug accumulates until [drug]_IN/[drug]_OUT = [H⁺]_IN/[H⁺]_OUT. As a proton is consumed by each neutral drug species, equilibrium is maintained by disassociation of methylammonium into methylamine and a proton. This accounts for the observed 1:1 molar stoichiometry between drug uptake and methylamine efflux.