ABSTRACT
Persistence of Mycobacterium tuberculosis is a hypoxia-inducible state in which the bacteria are phenotypically insensitive to currently available antituberculous drugs. In humans, persistent M. tuberculosis is found in granulomatous lesions, either inside macrophages or in necrotic tissue, where the partial oxygen pressure (pO(2)) is very low. Persistent bacteria can remain silent for decades before overt tuberculosis develops. Due to insensitivity to classical drugs, M. tuberculosis persistence prevents rapid and definitive clearance of bacteria. Consequently, therapeutic molecules are required that are both active against persistent bacilli and able to reach their intramacrophagic location. In contrast to its native form, norfloxacin is active in vivo against Mycobacterium bovis BCG present in the lungs when temporarily linked to a macromolecular carrier targeted to macrophages. To study the efficiency of this macromolecular prodrug targeted to persistent mycobacteria confined inside macrophages, we established a short-term in vivo model based on the physiological pO(2) differences between lungs, spleen and liver. Whereas lungs and spleen are well oxygenated, the liver has a low pO(2) due to its portal irrigation. Therefore, studying mycobacteria in the liver yields information about in vivo persistent bacilli exposed to low pO(2). To our knowledge, no similar short-term in vivo model has been published to date. Using this model, we demonstrated the insensitivity to isoniazid of M. bovis BCG present in hypoxic sites, and showed that norfloxacin given as a mannosylated macrophage-targeted prodrug was able to kill these isoniazid-insensitive mycobacteria. This demonstrates that intracellular persistent mycobacteria are amenable to antibiotic treatment.
Subject(s)
Antitubercular Agents/chemistry , Isoniazid/pharmacology , Liver/microbiology , Mycobacterium bovis/drug effects , Norfloxacin/chemistry , Animals , Antitubercular Agents/administration & dosage , Antitubercular Agents/pharmacology , Drug Design , Drug Resistance, Bacterial , Female , Hypoxia/physiopathology , Liver/physiopathology , Mice , Mice, Inbred C57BL , Molecular Structure , Norfloxacin/administration & dosage , Norfloxacin/pharmacology , Prodrugs , Spleen/microbiology , Spleen/physiopathologyABSTRACT
Macromolecular prodrugs of the antibiotic norfloxacin were prepared by coupling the drug via a peptide spacer onto a mannosylated dextran. The tetrapeptide gly-phe-gly-gly-gly-OMe was selected as substrate for lysosomal enzymes. The drug was coupled on the alpha-C of the terminal glycine. In vitro degradation studies demonstrated the release of the parent drug in the presence of cathepsin B. In vivo experiments on mice showed a promising therapeutic effect.
