Treatment of tuberculosis using a combination of sustained-release rifampin-loaded microspheres and oral dosing with isoniazid

Abstract

Previously, we reported on the use of rifampin-loaded microspheres to effectively treat Mycobacterium tuberculosis-infected macrophages and mice. Using similar biocompatible polymeric excipients of lactide and glycolide copolymers, we have increased the rifampin loading of small microsphere formulations (1 to 10 microm) by fourfold. Improved formulations were evaluated individually and in combination with oral regimens of isoniazid for the treatment of Mycobacterium tuberculosis H37Rv-infected mice. Groups (10 mice per group) consisted of mice that received (i) oral dosages of isoniazid (25 to 0.19 mg/kg of body weight/day), (ii) two intraperitoneal injections of rifampin-loaded microspheres on days 0 and 7, (iii) a combination of small rifampin-loaded microspheres on days 0 and 7 and isoniazid orally for 25 days (12.5 to 0.39 mg/kg/day), (iv) placebo injections, and (v) no treatment. Treatment with rifampin-loaded microspheres alone resulted in significant reductions in the numbers of CFU in the lungs and spleens by day 26. A bioassay revealed that plasma rifampin levels from the microspheres exceeded the MICs by more than twofold throughout the 26-day experimental period. Susceptibility testing demonstrated continued sensitivity to rifampin during the treatment period. Whereas isoniazid alone significantly reduced the numbers of CFU for dosages ranging from 12.5 to 1.56 mg/kg, combination therapy with rifampin-loaded microspheres increased the effective range to 0.39 mg/kg. In many cases, complete elimination of CFU was obtained with the combination therapy, something not achieved with most of the single therapies. These results demonstrate the ability to use small microsphere formulations alone to achieve significant results in a murine tuberculosis model and also the ability to use them safely in combination with another antimycobacterial agent.

FIG. 1

Synergistic interaction of rifamipin-loaded small microspheres (RIF) with orally administered isoniazid (INH) on mycobacterial CFU in the lungs of mice infected with M. tuberculosis H37Rv. The mice were infected and the tissues were processed for determination of the numbers of CFU as described in Materials and Methods. Results from experiment 1 (Table 1) are given as open symbols, and results from experiment 2 (Table 2) are given as closed symbols. Each point represents the mean for 10 mice, unless otherwise discussed in the text.

FIG. 2

Synergistic interaction of rifamipin-loaded small microspheres (RIF) with orally administered isoniazid (INH) on mycobacterial CFU in the spleens of mice infected with M. tuberculosis H37Rv. The mice were infected and the tissues were processed for determination of the numbers of CFU as described in Materials and Methods. Results from experiment 1 (Table 1) are given as open symbols, and results from experiment 2 (Table 2) are given as closed symbols. Each point represents the mean for 10 mice, unless otherwise discussed in the text.

FIG. 3

Release characteristics of small rifampin-loaded microspheres: lot 1 (5.8%; wt/wt) in experiment 1 and lot 2 (5.0%; wt/wt) in experiment 2. Mice were randomly chosen from experimental group 1, which received the 58-mg (○) and 116-mg (•) doses or experimental group 2, which received the 50-mg (▫) and 100-mg (▪) doses. At days 7, 14, and 21, blood was obtained from three mice by retro-orbital bleeding. At the conclusion of the experiment (day 26), all 10 mice in each of the experimental groups were exsanguinated by cardiac puncture. Plasma was assayed by using S. aureus, as described in Materials and Methods. Each point represents the mean for either 3 (days 7, 14, and 21) or 10 (day 26) assayed samples. The upper MIC for M. tuberculosis H37Rv is given as a dotted line (0.25 μg/ml) and corresponds to the upper value of the MIC range discussed in Materials and Methods (0.06 to 0.25 μg/ml).