Pharmacodynamics of Amoxicillin/clavulanic Acid Against Haemophilus Influenzae in an in Vitro Kinetic Model: a Comparison of Different Dosage Regimens Including a Pharmacokinetically Enhanced Formulation

Objective: To study the pharmacodynamics of amoxicillin/clavulanic acid against different strains of Haemophilus influenzae in an in vitro kinetic model. The concentrations used corresponded to human serum levels obtained after 875 mg amoxicillin/clavulanic acid given b.i.d., 500/125 mg amoxicillin/clavulanic acid given t.i.d. and those obtained with a pharmacokinetically enhanced formulation containing 1125/125 mg amoxicillin/clavulanic acid (immediate release) and 875 mg amoxicillin (sustained release) given b.i.d.

Methods: Bacteria at an initial inoculum of 106 colony-forming units (CFU)/mL were exposed to amoxicillin/clavulanic acid with an initial concentration of approximately 15/3 mg/L, 8/3 mg/L simulating the peak levels in humans achieved after a dose of 875/125 mg and 500/125 mg with a half-life of 1 h. In addition, experiments with a 2000/125 mg pharmacokinetically enhanced formulation of amoxicillin/clavulanic acid given b.i.d. were performed. A repeated dose was given at 12 h after the initial dose of 875/125 mg and the pharmacokinetically enhanced formulation or at 8 and 16 h after the dose of 500/125 mg. The experiments were performed in an in vitro kinetic model, which consists of a spinner flask with a filter membrane fitted in between the upper part and the bottom part in order to prevent bacterial dilution. The medium is removed from the culture flask, through the filter, at a constant rate with a pump. Repeated samples were taken at intervals of 1-2 h up to 24 h during the experiments for viable counting. One of the strains of H. influenzae was also exposed to a constant concentration corresponding to the peak serum levels obtained after a dose of 500/125 mg.

Results: The concentrations of amoxicillin in the in vitro kinetic model were as expected. At the end of the experiment (24 h), there was a tendency for a greater bactericidal effect with 500/125 mg t.i.d., as compared to 875/125 b.i.d., with differences in CFUs between the two dosing regimens of 2.6 log10 CFU for H. influenzae LH 2803 and 1.8 log10 CFU for the other clinical strains. However, these differences did not reach statistical significance (P = 0.075 and 0.10, respectively). A statistically significant higher bactericidal effect was seen in the experiments with the pharmacokinetically enhanced formulation in comparison with the b.i.d. regimen both at 8, 16 and 24 h and at 8 and 16 h with the t.i.d. regimen. With the new formulation, no regrowth was seen at 24 h, similar to the results obtained with a constant concentration.

Conclusions: Neither of the standard dosing regimens of amoxicillin (875/125 mg b.i.d. or 500/125 mg) used in our study, in which the time that the free (non-protein-bound) concentration the MIC (T > MIC) exceeding was less than 50%, was sufficient to achieve a complete bactericidal effect during the first 24 h of treatment. However, a statistically significant difference in bactericidal activity was seen at 8, 16 and 24 h vs. the b.i.d. regimen and at 8 and 16 h vs. the t.i.d. regimen with the pharmacokinetically enhanced formulation. This formulation gave a longer T > MIC (73-79%) of amoxicillin even though the concentration of clavulanic acid was only detectable for 45% of the dosing interval, and complete killing of all strains was obtained after 24 h.