The preferential localization of liposomes at sites of infection offers an attractive way to selectively increase antibacterial drug concentrations at the target location, with the intention to increase the therapeutic efficacy of antimicrobial treatment. Most studies in this field have been performed in animal models with an intact host defense and with high-antibiotic-susceptible bacteria. However, in clinical practice, two important complicating factors should be taken into account. In particular, patients having impaired host defenses carry a high risk of treatment failure, which further increases when the infectious organism has moderate to low susceptibility to the applied antimicrobial agent. Generally, the studies with animals with intact host defenses report enhanced therapeutic efficacy of the liposomal formulation compared to that of the free drug (3
). The same conclusion can be drawn from the present study in the intact host defense model of a high-GEN-susceptible K. pneumoniae
pneumonia. As a result of the local delivery of the antibiotic by LE-GEN, a single dose of the liposome-encapsulated drug dramatically improves survival compared to an equivalent dose of free GEN. Increasing the dose of LE-GEN may improve survival to 100%. Yet the clinical relevance of this approach is limited, as a relatively short course of treatment for only 3 days with free GEN at 5 mg/kg/day q12h already yields complete survival. Therefore, the present study was undertaken to investigate the therapeutic potential of LE-GEN in clinically more relevant models of infection that are difficult to treat with conventional antibiotics as a result of impaired host defense and low antibiotic susceptibility of the inoculated bacteria.
In leukopenic rats inoculated with the high-susceptible K. pneumoniae strain, approximately 10 to 100 bacteria per ml of blood were already present 24 h after injection. Thus, in these leukopenic rats, antimicrobial therapy should be directed not only towards the bacteria at the infectious focus (the left lung) but also towards the rapidly occurring bacteremia. The survival experiments in the leukopenic model of a high-GEN-susceptible K. pneumoniae pneumonia show that antimicrobial treatment is far less effective as a result of impaired host defense. Treatment with free GEN at 5 mg/kg/day q12h for 3 days, which produced complete survival in the rats with intact host defenses, hardly prolongs survival in the leukopenic animals. Continuing treatment up to 5 days prolonged survival during treatment. However, after termination of treatment, only 10% survived up to 14 days. Doses of free GEN have to be increased up to 40 mg/kg/day for 5 days (the MTD) to obtain complete survival. These data illustrate why in clinical practice aminoglycosides are used in combination with other classes of antibiotics to increase the therapeutic efficacy under these conditions.
Addition of a single dose of LE-GEN at 5 mg/kg on day 1 to the 5-day treatment with free GEN at 5 mg/kg/day q12h appeared to confer substantial therapeutic benefit. All rats survived, and the cumulative amount of GEN administered was sevenfold lower than the amount of free GEN (40 mg/kg/day for 5 days) needed to obtain complete survival. This reduction in GEN exposure may reduce the risk of the well-known toxicity of GEN on the kidney and audiovestibular apparatus. On the other hand, the altered tissue distribution in general and increased GEN concentrations at the site of infection in particular as a result of the liposome encapsulation might change the toxicity profile. Yet previous studies in beagle dogs with liposome-encapsulated amikacin suggest a favorable safety profile for liposome-encapsulated aminoglycosides, as doses of 20 mg/kg/day for 1 month did not result in adverse effects despite steady-state plasma concentrations of 750 μg/ml (14
). Rats treated with only LE-GEN at 5 mg/kg for 5 days already show mortality during treatment. The blood clearance kinetics of free GEN and LE-GEN offer an explanation for the superior efficacy of the combination of free GEN and LE-GEN. Free GEN is therapeutically active in the circulation against the bacteremia but is rapidly cleared after injection, with a half-life of approximately 20 min. Activity in the infected left lung is expected to be limited. When encapsulated in liposomes, on the other hand, the drug only slowly leaves the circulation. LE-GEN hardly releases GEN into the bloodstream and consequently shows limited activity against bacteremia but localizes substantially in the infected left lung (3
). Release of GEN from the liposomes localizing in the infected lung leads to the efficient bacterial killing seen. As a result, combination of free GEN and LE-GEN reduces the numbers of bacteria in left-lung tissue and blood 10- and 100-fold more efficiently, respectively, than the treatment with free GEN alone. These numbers were further reduced to zero in the remaining study period. In contrast, the majority of rats treated with free GEN or LE-GEN alone died during this period. The third model addresses an additional factor complicating clinical antimicrobial therapy, i.e., low antibiotic susceptibility of the bacteria. The eightfold increase in the MIC clearly had an effect on the efficacy of treatment, as free GEN at the MTD was no longer effective and all rats died. The addition of LE-GEN at 20 mg/kg/day q24h for 5 days to the 5-day treatment with free GEN at the MTD resulted in 50% survival on day 14 without producing acute toxicity. Probably, as a result of the low GEN susceptibility of the K. pneumoniae
strain, the therapeutic availability of GEN released from this rigid liposome formulation in the infected lung is insufficient to obtain 100% survival. As fluid liposomes have been reported to release encapsulated aminoglycosides more easily than their rigid counterparts (7
), a fluid LE-GEN formulation was investigated. Cholesterol was omitted from the liposome formulation, as cholesterol has a major rigidifying effect on liposomal bilayers (10
). Furthermore, the partially hydrogenated phospholipid PHEPC was replaced by EPC as bilayer rigidity increases with the degree of hydrogenation of the phospholipids in the bilayer. The addition of fluid LE-GEN instead of rigid LE-GEN to 5-day free-GEN treatment at the MTD resulted in an increase in therapeutic effect: complete survival was obtained. The blood clearance kinetics of fluid LE-GEN show that GEN is released into the circulation to a larger extent from this liposome formulation than from rigid LE-GEN. As a result, higher therapeutically active drug concentrations were measured in the bloodstream. Yet, a sufficient amount of GEN remained liposomally encapsulated to be delivered to the left lung to control the local infection, as is supported by the efficient bacterial killing observed in the left lung. These results show that lipid composition is an important determinant of therapeutic efficacy. A careful balance must be sought between release of antibiotic into the circulation to obtain sufficiently high drug levels there versus liposomal retention of the drug in order to achieve sufficiently high levels of (locally released) antibiotic at the infectious focus.
In conclusion, in rats with intact host defenses infected with a high-GEN-susceptible K. pneumoniae strain, LE-GEN is clearly superior to free GEN treatment. Yet the clinical relevance is limited, as complete survival can also be obtained with multiple doses of the free drug. In leukopenic rats infected with high-GEN-susceptible K. pneumoniae cells, the addition of LE-GEN to free GEN treatment shows substantial therapeutic benefit. Complete survival can be obtained by using a sevenfold-lower amount of GEN compared to administration of free GEN alone. In leukopenic rats infected with low-GEN-susceptible K. pneumoniae cells, free GEN at the MTD shows 0% survival. The use of LE-GEN is a strict requirement for achieving therapeutic success. It appears that the increased release of GEN by fluid LE-GEN compared to rigid LE-GEN is more favorable. These results warrant further clinical studies of liposomal formulations of aminoglycosides in immunocompromised patients with severe infections.