JAC Advance Access originally published online on January 22, 2007
Journal of Antimicrobial Chemotherapy 2007 59(3):499-506; doi:10.1093/jac/dkl533
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Uptake of inhalable microparticles affects defence responses of macrophages infected with Mycobacterium tuberculosis H37Ra
1 Pharmaceutics Division, Central Drug Research Institute, Chhattar Manzil Palace, Lucknow 226001, India 2 Toxicology Division, Central Drug Research Institute, Chhattar Manzil Palace, Lucknow 226001, India 3 EM Unit, Central Drug Research Institute, Chhattar Manzil Palace, Lucknow 226001, India 4 Inhalation Toxicology Division, Industrial Toxicology Research Centre, Gheru, Lucknow 226001, India
Received 31 August 2006; returned 26 October 2006; revised 4 December 2006; accepted 10 December 2006
* Corresponding author. Tel: +91-522-261-2411, ext. 4302; Fax: +91-522-261-4305; E-mail: amit.cdri{at}gmail.com
Objectives: To investigate whether inhalable microparticles containing two anti-tuberculosis agents, isoniazid and rifampicin, evoke host-defence strategies in macrophages in addition to targeting the incorporated drugs.
Methods: Microparticles were prepared by spray-drying a homogeneous solution of drugs and poly(lactic acid) (PLA; apparent viscosity 1.1 cP). Four parts PLA and three parts rifampicin were dissolved in dichloromethane. One part isoniazid was dissolved in methanol. The two solutions were mixed in the ratio 22 : 3 at which none of the solutes precipitated. These were administered as ‘nose-only’ inhalations to mice or exposed to cultured J774 mouse macrophages. Targeting to lung macrophages was investigated by transmission electron microscopy. Reactive oxygen species (ROS) were estimated by a cytochrome c assay and flow cytometry. Reactive nitrogen intermediates (RNI) were assayed using Griess reagent. Cytokines in culture supernatants were estimated by ELISA.
Results: Treatment with inhalable microparticles targeted lung macrophages in vivo and induced intense Golgi activity in the vicinity of microparticle-containing phagosomes. Microparticles induced a respiratory burst involving NADPH oxidase and enhanced NO production by infected macrophages. Microparticle-induced NADPH oxidase activation required optimal calcium ions. Microparticles efficiently induced tumour necrosis factor-
(TNF-) secretion by macrophages recovered from infected mice.
Conclusions: Microparticle phagocytosis induces responses in infected murine macrophages that are indicative of activation of innate bactericidal mechanisms, and are inimical to bacterial survival. It is likely that such responses augment straightforward drug action on the bacterium and contribute to the unexpectedly high efficacy of microparticles in experimental tuberculosis.
Keywords: biodegradable microspheres , targeting , tuberculosis , innate immune response , free radicals , cytokines
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