Abstract
ObjectivesMycobacterium avium complex (MAC) bacteria can cause chronic pulmonary disease (PD). Current treatment regimens of azithromycin, ethambutol and rifampicin have culture conversion rates of around 65%. Dynamic, preclinical models to assess the efficacy of treatment regimens are important to guide clinical trial development. The hollow fibre system (HFS) has been applied but reports lack experimental details. MethodsWe simulated the human pharmacokinetics of azithromycin, ethambutol and rifampicin both in plasma and epithelial lining fluid (ELF) in a HFS, exposing THP-1 cells infected with M. avium to the triple-drug regimen for 3 weeks. We accounted for drug–drug interactions and protein-binding and provide all laboratory protocols. We differentiated the effects on the intracellular and extracellular mycobacterial population. ResultsThe antibiotic concentrations in the HFS accurately reflected the time to peak concentration (Tmax), the peak concentration (Cmax) and half-life of azithromycin, rifampicin and ethambutol in plasma and ELF reported in literature. We find that plasma drug concentrations fail to hold the MAC bacterial load static (ΔLog10 CFU/mLControl:Regimen = 0.66 ± 0.76 and 0.45 ± 0.28 at 3 and 21 days); ELF concentrations do hold the bacterial load static for 3 days and inhibit bacterial growth for the duration of the experiment (ΔLog10 CFU/mLControl:Regimen = 1.1 ± 0.1 and 1.64 ± 0.59 at 3 and 21 days). DiscussionIn our model, the current therapy against MAC is ineffective, even when accounting for antibiotic accumulation at the site of infection and intracellularly. New treatment regimens need to be developed and be compared with currently recommended regimens in dynamic models prior to clinical evaluation. With the publication of all protocols we aim to open this technology to new users.
Highlights
The hollow fibre system (HFS) has been used to search for new MAC-PD regimens, but previous studies lacked experimental detail and have failed to address four key issues: (a) to simulate the currently recommended azithromycineethambutolerifampicin regimen as a point of reference; (b) to simulate the pharmacokinetics in blood plasma and in lung epithelial lining fluid (ELF), which is more representative of the site of infection; (c) to account for protein binding of the drugs in these matrices [7e10]; and (d) to simulate drug penetration into immune cells such as macrophages
Azithromycin, ethambutol and rifampicin were purchased from Sigma Aldrich (Zwijndrecht, the Netherlands) Mycobacterium avium ATCC 700898 reference strain was acquired from ATCC (Molsheim Cedex, France) and freshly cultured before each experiment; the minimum inhibitory concentrations of study drugs were determined by broth microdilution according to CLSI guidelines [11]
Half-lives and Tmax of ethambutol and azithromycin were obtained from Magis-Escurra et al and van Ingen et al As no full pharmacokinetic curves are available for ELF concentrations, we extrapolated these from available data
Summary
In vitro studies of MAC-PD treatment often expose planktonic bacteria to a multiplicity of the minimum inhibitory concentration of antibiotics, alone or together with companion drugs [5,6] but this approach does not mimic the dynamic drug exposures at the site of infection in the context of a multidrug regimen. The HFS has been used to search for new MAC-PD regimens, but previous studies lacked experimental detail and have failed to address four key issues: (a) to simulate the currently recommended azithromycineethambutolerifampicin regimen as a point of reference; (b) to simulate the pharmacokinetics in blood plasma and in lung epithelial lining fluid (ELF), which is more representative of the site of infection; (c) to account for protein binding of the drugs in these matrices [7e10]; and (d) to simulate drug penetration into immune cells such as macrophages
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