Koji Takemoto

Evaluation of Antifungal Pharmacodynamic Characteristics of AmBisome against Candida albicans

A liposomal formulation of Amphotericin B (AmBisome), with small unilamellar vesicles containing amphotericin B, shows characteristic pharmacokinetics as liposomes, and in consequence, has different pharmacological activity and toxicity from amphotericin B deoxycholate (Fungizone). In this study, we evaluated the antifungal pharmacodynamic characteristics of AmBisome against Candida albicans using the in vitro time‐kill method and murine systemic infection model. A time‐kill study indicated that the in vitro fungicidal activities of AmBisome and Fungizone against C. albicans ATCC 90029 increased with increasing drug concentration. For in vivo experiments, leucopenic mice were infected intravenously with the isolate 4 hr prior to the start of therapy. The infected mice were treated for 24 hr with twelve dosing regimens of AmBisome administered at 8‐, 12‐, 24‐hr dosing intervals. Correlation analysis between the fungal burden in the kidney after 24 hr of therapy and each pharmacokinetic/pharmacodynamic parameter showed that the peak level/MIC ratio was the best predictive parameter of the in vivo outcome of AmBisome. These results suggest that AmBisome, as well as Fungizone, has concentration‐dependent antifungal activity. Furthermore, since AmBisome can safely achieve higher concentrations in serum than Fungizone, AmBisome is thought to have superior potency to Fungizone against fungal infections.

Influence of the Progression of Cryptococcal Meningitis on Brain Penetration and Efficacy of AmBisome in a Murine Model

Background: AmBisome is a small unilamellar vesicle containing amphotericin B. AmBisome is generally unable to pass through the blood-brain barrier, but the distribution of AmBisome in the brain is increased by inflammation, and in consequence, AmBisome exhibits activity against fungal meningitis. We investigated the influence of the progression of cryptococcal meningitis on the brain penetration and efficacy of AmBisome. Method: Mice were infected intracerebroventricularly with Cryptococcus neoformans 4 h or 5 days prior to a single dose treatment. Results: The brain tissue level and efficacy of AmBisome when administered 5 days after infection were greater than 4 h after infection. An immunohistochemical study showed that AmBisome-derived amphotericin B was localized at the infected site in the subarachnoid space. When AmBisome was compared with Fungizone at the maximum tolerated dose, 10 mg/kg AmBisome exhibited greater efficacy than 1 mg/kg Fungizone in both regimens. Conclusion: The brain penetration of AmBisome was enhanced by the progression of cryptococcal meningitis and correlated with the in vivo activity.

Temperature-dependent transfer of amphotericin B from liposomal membrane of AmBisome to fungal cell membrane

Liposomal amphotericin B (AMPH-B), also known as AmBisome, exhibits a potent antifungal effect through its binding to ergosterol contained within the fungal cell membrane. However, the mechanism responsible for the movement of AmBisome-derived AMPH-B to the fungal cell membrane through the cell wall is not yet clear. Therefore, in the present study we aimed at elucidating this mechanism operating in Saccharomyces cerevisiae. AmBisome showed its antifungal effect against S. cerevisiae at 35 degrees C but not at 4 degrees C, whereas free AMPH-B was effective at both temperatures. A significant difference in the amount of AMPH-B transferred to the fungal cells between incubation at 4 and 35 degrees C was also observed when AmBisome was used. Confocal microscopic study, however, indicated that NBD-labeled AmBisome was localized on the surface of the fungal cells at either temperature. To decrease the affinity of AMPH-B for the liposomal membrane, we entrapped AMPH-B in fluid liposomes containing egg yolk phosphatidylcholine (EPC) instead of hydrogenated soy PC (HSPC). These liposomes showed the antifungal effect even at 4 degrees C. On the contrary, AMPH-B in liposomes containing ergosterol (Erg-AmB) instead of cholesterol showed a significantly weaker antifungal effect at 35 degrees C with reduced transfer of AMPH-B to the fungal cells. These results suggest that not the binding of AmBisome to target cells but the transfer of AMPH-B from liposomal membrane of AmBisome to the cell membrane is critical for the antifungal activity of AmBisome. This transfer is dependent on the temperature, fluidity of the liposomal membrane, and the affinity of AMPH-B for the fungal cell membrane.

Novel Carbapenem Antibiotics for Parenteral and Oral Applications: In Vitro and In Vivo Activities of 2-Aryl Carbapenems and Their Pharmacokinetics in Laboratory Animals

ABSTRACT SM-295291 and SM-369926 are new parenteral 2-aryl carbapenems with strong activity against major causative pathogens of community-acquired infections such as methicillin-susceptible Staphylococcus aureus, Streptococcus pneumoniae (including penicillin-resistant strains), Streptococcus pyogenes, Enterococcus faecalis, Klebsiella pneumoniae, Moraxella catarrhalis, Haemophilus influenzae (including β-lactamase-negative ampicillin-resistant strains), and Neisseria gonorrhoeae (including ciprofloxacin-resistant strains), with MIC90s of ≤1 μg/ml. Unlike tebipenem (MIC50, 8 μg/ml), SM-295291 and SM-369926 had no activity against hospital pathogens such as Pseudomonas aeruginosa (MIC50, ≥128 μg/ml). The bactericidal activities of SM-295291 and SM-369926 against penicillin-resistant S. pneumoniae and β-lactamase-negative ampicillin-resistant H. influenzae were equal or superior to that of tebipenem and greater than that of cefditoren. The therapeutic efficacies of intravenous administrations of SM-295291 and SM-369926 against experimentally induced infections in mice caused by penicillin-resistant S. pneumoniae and β-lactamase-negative ampicillin-resistant H. influenzae were equal or superior to that of tebipenem and greater than that of cefditoren, respectively, reflecting their in vitro activities. SM-295291 and SM-369926 showed intravenous pharmacokinetics similar to those of meropenem in terms of half-life in monkeys (0.4 h) and were stable against human dehydropeptidase I. SM-368589 and SM-375769, which are medoxomil esters of SM-295291 and SM-369926, respectively, showed good oral bioavailability in rats, dogs, and monkeys (4.2 to 62.3%). Thus, 2-aryl carbapenems are promising candidates that show an ideal broad spectrum for the treatment of community-acquired infections, including infections caused by penicillin-resistant S. pneumoniae and β-lactamase-negative ampicillin-resistant H. influenzae, have low selective pressure on antipseudomonal carbapenem-resistant nosocomial pathogens, and allow parenteral, oral, and switch therapies.

Comparative Study on the Efficacy of Liposomal Amphotericin B and Voriconazole in a Murine Pulmonary Aspergillosis Model

Background:No clinical studies have compared the efficacy of liposomal formulation AMB (L-AMB) and voriconazole (VRC) in the treatment of pulmonary aspergillosis. The aim of this study was to compare the efficacy of L-AMB and VRC in murine pulmonary aspergillosis.Methods: Leucopenic mice were infected intratracheally with Aspergillus fumigatus and treated intravenously with L-AMB (once a day) or VRC (twice a day). Results: L-AMB and VRC at a dose of ≥5 and ≥20 mg/kg, respectively, significantly prolonged the survival time of infected mice and reduced the pulmonary fungal burden in comparison with the control group. At the maximum recommended dose for clinical use, 5 mg/kg of L-AMB exhibited greater efficacy than 10 mg/kg of VRC, which achieved an area under the concentration-time curve level equivalent to that of 6 mg/kg (loading dose) in humans, in terms of increasing survival and reducing the fungal burden. Conclusion: The in vivo efficacy of L-AMB was superior to that of VRC at the maximum recommended dose in a murine pulmonary aspergillosis model.

AmBisome: relationship between the pharmacokinetic characteristics acquired by liposomal formulation and safety/efficacy

Abstract Amphotericin B (AMPH-B) is a polyene antifungal agent with a superior and broad fungicidal spectrum, but its administration at a dose sufficient for treatment is difficult because of dose- and duration-dependent nephrotoxicity. To solve this dilemma, a liposomal formation of AMPH-B that achieved reduction of adverse effects while maintaining the efficacy, AmBisome® (L-AMB), was developed. In L-AMB, AMPH-B molecules are stabilized by phospholipids and cholesterol in the liposomal lipid bilayer, reducing the cytotoxicity for animal cells compared with that of the free-form AMPH-B. In addition, extravascular permeation of L-AMB is limited in normal tissue because of the liposome particle size (particle diameter: 100 nm or smaller), a high blood level is maintained and the distribution in organs, including the kidney is reduced, contributing to the improvement of the safety. On the other hand, vascular permeation increases due to inflammation and damage by fungal invasion, which increases L-AMB transfer from the circulation to lesions and its antifungal activity. Furthermore, since the parameter most closely correlated with the in vivo outcome of L-AMB is Cmax/MIC, the pharmacokinetic (PK) characteristics of L-AMB, which result in a high blood level than that for the free-form AMPH-B, is advantageous for antifungal activity. Incorporation of AMPH-B into the liposomal membrane resulted in PK characteristics of L-AMB markedly different from those of AMPH-B, and the superior efficacy and safety were achieved based on these characteristics. In this review, the relationship between the PK characteristics of L-AMB and safety/efficacy is introduced.

Influence of Fungicidal Activity against Candida tropicalis on the Efficacy of Micafungin and Liposomal Amphotericin B in a Neutropenic Murine Lethal Infection Model

Objectives: To investigate the correlation between in vitro killing activity and in vivo efficacy of micafungin (MCFG) and liposomal amphotericin B (L-AMB) against Candida tropicalis in a neutropenic murine lethal infection model. Methods:Candida albicans (one strain) and C. tropicalis (three strains) were tested in time-kill studies. Cyclophosphamide-treated mice were inoculated intravenously with each strain. One day after inoculation, antifungals were administered intravenously once daily for 1 or 3 days. Results: MCFG exhibited fungicidal activity against C. albicans ATCC 90029 and C. tropicalis SP-20142, and fungistatic activity against C. tropicalis ATCC 42678 and SP-20047. The ED50s (dosage that results in 50% survival) of MCFG for C. tropicalis ATCC 42678 and SP-20047 (4.1–50 mg/kg) were higher than those for other strains (1.6–12 mg/kg). A 1-day course of MCFG was not effective against C. tropicalis ATCC 42678 and SP-20047 at the clinical dose (5 mg/kg), which achieved an AUC level almost equal to that of 100 mg in humans, whereas a 3-day course of 5 mg/kg MCFG was efficacious against all strains. In contrast, L-AMB showed fungicidal activity against all strains tested and the ED50s of L-AMB were 0.08–0.65 mg/kg. In both treatment regimens, the minimum effective doses of L-AMB (≤0.5 mg/kg) were less than the clinical dosage (≤5 mg/kg). Conclusions: The in vivo efficacy of MCFG and L-AMB showed a correlation with the in vitro killing activity. At the clinical dose, L-AMB exerted anti-C. tropicalis activity within a shorter treatment period than MCFG.

Efficacy of High-Dose Meropenem (Six Grams per Day) in Treatment of Experimental Murine Pneumonia Induced by Meropenem-Resistant Pseudomonas aeruginosa

ABSTRACT High-dose meropenem (MEPM; 6 g/day) has been approved as a treatment for purulent meningitis; however, little is known regarding its in vivo efficacy in refractory lower respiratory tract infections. The purpose of this study was to evaluate the efficacy of MEPM at 6 g/day in a murine model of severe pneumonia caused by MEPM-resistant Pseudomonas aeruginosa. Experimental pneumonia induced by MEPM-resistant P. aeruginosa was treated with normal-dose MEPM (150 mg/kg of body weight, simulating a 3-g/day regimen in humans) or high-dose MEPM (500 mg/kg, simulating a 6-g/day regimen in humans). Mice treated with high-dose MEPM showed significantly restored survival relative to that of untreated mice and tended to show a survival rate higher than that of mice treated with normal-dose MEPM. The viable bacterial counts (of two clinical isolates) in the lungs decreased significantly in mice treated with high-dose MEPM from those for untreated mice (P < 0.001) or mice treated with normal-dose MEPM (P, <0.01 and <0.05). The number of inflammatory cells in the bronchoalveolar lavage fluid (BALF) was also significantly lower in mice treated with high-dose MEPM than in untreated mice. The free MEPM concentration in the epithelial lining fluid (ELF) exceeded 16 μg/ml for 85 min in mice treated with high-dose MEPM, but not for mice treated with normal-dose MEPM. Our results demonstrate that high-dose MEPM (6 g/day) might provide better protection against pneumonia caused by MEPM-resistant strains of P. aeruginosa than the dose normally administered (less than 3 g/day).