Tissue Distribution of Doxycycline in Animal Models of Tuberculosis

Abstract

Doxycycline, an FDA-approved tetracycline, is used in tuberculosis <i>in vivo</i> models for the temporal control of mycobacterial gene expression. In these models, animals are infected with recombinant <i>Mycobacterium tuberculosis</i> carrying genes of interest under transcriptional control of the doxycycline-responsive TetR-<i>tetO</i> unit. To minimize fluctuations of plasma levels, doxycycline is usually administered in the diet. However, tissue penetration studies to identify the minimum doxycycline content in food achieving complete repression of TetR-controlled genes in tuberculosis (TB)-infected organs and lesions have not been conducted. Here, we first determined the tetracycline concentrations required to achieve silencing of <i>M. tuberculosis</i> target genes <i>in vitro</i> Next, we measured doxycycline concentrations in plasma, major organs, and lung lesions in TB-infected mice and rabbits and compared these values to silencing concentrations measured <i>in vitro</i> We found that 2,000 ppm doxycycline supplemented in mouse and rabbit feed is sufficient to reach target concentrations in TB lesions. In rabbit chow, the calcium content had to be reduced 5-fold to minimize chelation of doxycycline and deliver adequate oral bioavailability. Clearance kinetics from major organs and lung lesions revealed that doxycycline levels fall below concentrations that repress <i>tet</i> promoters within 7 to 14 days after doxycycline is removed from the diet. In summary, we have shown that 2,000 ppm doxycycline supplemented in standard mouse diet and in low-calcium rabbit diet delivers concentrations adequate to achieve full repression of <i>tet</i> promoters in infected tissues of mice and rabbits.