Tissue Distribution of Doxycycline in Animal Models of Tuberculosis

Abstract

Doxycycline, an FDA-approved tetracycline, is used in tuberculosis in vivo models for the temporal control of mycobacterial gene expression. In these models, animals are infected with recombinant Mycobacterium tuberculosis carrying genes of interest under transcriptional control of the doxycycline-responsive TetR-tetO 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 M. tuberculosis target genes in vitro 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 in vitro 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 tet 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 tet promoters in infected tissues of mice and rabbits.

Keywords: TetR; animal models; doxycycline; genetic regulation in vivo; tissue penetration; tuberculosis.

FIG 1

Effect of tetracyclines on in vitro growth of M. tuberculosis H37Rv harboring essential genes under TetR control compared to wild type. (A and B) Growth inhibitory dose response of anhydrotetracycline (ATc) and doxycycline (DOX) in M. tuberculosis strains that harbor a TetR-dependent dual-control genetic switch (14) driving expression of Fum (fumarate hydratase, green symbols), Rho (transcription termination factor, orange symbols), or TrxB2 (thioredoxin reductase, blue symbols). (C) Growth inhibitory dose response of ATc and DOX against wild-type M. tuberculosis H37Rv.

FIG 2

Plasma and tissue pharmacokinetics of DOX in mice receiving DOX-supplemented diet. (A) Box and whisker plot of DOX plasma concentrations in mice receiving 2,000 ppm DOX in standard diet for 7 days. Mean values (bar), minimum and maximum (box) and standard deviations (error bars) are shown for n = 4 to 11 mice per group. The orange shaded window indicates the range of in vitro DOX concentration that inhibits 90% of bacterial growth (IC₉₀) for the three reporter strains shown in Fig. 1. (B) DOX concentrations in mouse organs relative to plasma after 7 days on the DOX diet. Mean values and standard deviations (error bars) are shown (n = 3). (C) Kinetics of DOX clearance in plasma following removal of DOX from the diet for 14 days. Mean values (bar) and minimum and maximum (box) and standard deviations (error bars) are shown (n = 3 to 12). The orange shaded window indicates the range of in vitro DOX concentrations that inhibit 50% of bacterial growth (IC₅₀) for the three reporter strains shown in Fig. 1. (D) Kinetics of DOX clearance from major organs of TB-infected mice 7 and 14 days after removal of DOX from the diet. Day 1 corresponds to DOX levels in mice that had received a DOX-supplemented diet for 1 week, after which it was replaced by a DOX-free diet for the duration of the experiment. The purple dotted line indicates the lower boundary of published MIC ranges against wild-type M. tuberculosis (11), and the black dotted lines show individual DOX IC₅₀s for the three reporter strains (Fig. 1). Mean values and standard deviations (error bars) are shown (n = 3).

FIG 3

Plasma and tissue pharmacokinetics of DOX in rabbits receiving DOX-supplemented chow. (A) Left panel: DOX concentration-time profile in rabbit plasma following a single DOX dose administered intravenously (i.v.), by oral gavage (p.o.), and subcutaneously (s.c.), as indicated. Right panel: DOX plasma concentrations in rabbits receiving 2,000 ppm DOX in standard chow for 7 days. AM, immediately prior to receiving daily DOX-supplemented feed in the morning; PM, 6 h after receiving DOX feed. (B) Effect of dietary calcium removal on the plasma pharmacokinetics of DOX in rabbits following a single oral dose of 25 mg/kg. (C) Effect of dietary calcium lowering (0.2% total calcium content, or 5-fold lower than in standard chow) on DOX plasma levels in rabbits that received 2,000 ppm DOX in chow for 5 to 7 days. AM and PM time points are as described for right panel A. Data are represented as scatter dot plots of individual animal values and are pooled from two separate experiments with 4 and 6 rabbits, respectively (horizontal bar indicates mean, n = 20 to 34 blood samples from 10 rabbits).

FIG 4

DOX penetration in major organs, lung lesions, and necrotic caseum of TB-infected rabbits. (A) DOX concentrations in plasma, uninvolved lung tissue, lung lesions, and major organs in 4 rabbits receiving a low-calcium 2,000 ppm DOX diet for 7 days. Mean values and standard deviations (error bars) are shown (n = 3). (B) Scatter dot plots of caseum-to-cellular DOX concentration ratios in necrotic granulomas collected from TB-infected lungs. Ratios obtained in 10 lesions from 5 infected rabbits are shown.