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

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.

Exposure of human peripheral blood mononuclear cells to total lipids and serovar-specific glycopeptidolipids from Mycobacterium avium serovars 4 and 8 results in inhibition of TH1-type responses.

Previous studies have suggested that large quantities of bacterial lipids may accumulate and persist within host cells during chronic stages of Mycobacterium avium infections. This study intended to assess the ability of purified M. avium lipids to affect TH-1-type responses in human peripheral blood mononuclear cells (PBMC) from healthy donors. PBMC were exposed to total lipids and serovar-specific glycopeptidolipids (GPL) extracted from M. avium serovars 4 and 8, which have been reported to predominate as opportunistic infection among AIDS patients. After 24 h exposure to lipids followed by PHA/PMA treatment, IL-2 and IFN-gamma were assayed in the supernatants. Reverse transcriptase polymerase chain reaction (RT-PCR) was used for a semiquantitative estimation of mRNA for IL-2 and IFN-gamma in cell pellets at various time points. Exposure of PBMC to M. avium total lipids significantly suppressed PHA/PMA-induced secretion of IL-2 and IFN-gamma as determined by ELISA. The GPL antigens from serovar 4 were more efficient at inhibiting TH-1 responses than GPL from serovar 8. CD4(+)T-lymphocyte enrichment of PBMC demonstrated that suppression by M. avium lipids was intact without the presence of other cell populations such as monocytes and B-cells. Preliminary RT-PCR experiments showed that the secretion of TH-1 cytokines was partially affected at the transcriptional level. The results obtained showed that M. avium lipids are indeed able to modify the induction of TH-1-type cytokines by human PBMC, and suggest that accumulation of M. avium lipids in the chronic stages of infection may play an important role in the pathogenesis of HIV infection.

Efficacy of microencapsulated rifampin in Mycobacterium tuberculosis-infected mice.

Rifampin is a first-line drug useful in the treatment of tuberculosis. By using biocompatible polymeric excipients of lactide and glycolide copolymers, two microsphere formulations were developed for targeted and sustained delivery of rifampin, with minimal dosing. A small-microsphere formulation, with demonstrated ability to inhibit intracellularly replicating Mycobacterium tuberculosis H37Rv, was tested along with a large-microsphere formulation in an infected mouse model. Results revealed that by using a single treatment of the large-microsphere formulation, it was possible to achieve a significant reduction in M. tuberculosis H37Rv CFUs in the lungs of mice by 26 days postinfection. A combination of small (given as two injections on day 0 and day 7) and large (given as one injection at day 0) rifampin-loaded microsphere formulations resulted in significant reductions in CFUs in the lungs by 26 days, achieving a 1.23 log10 reduction in CFUs. By comparison, oral treatment with 5, 10, or 20 mg of rifampin/kg of body weight, administered every day, resulted in a reduction of 0.42, 1.7, or 1.8 log10 units, respectively. Thus the microsphere formulations, administered in one or two doses, were able to achieve results in mice similar to those obtained with a daily drug regimen within the range of the highest clinically tolerated dosage in humans. These results demonstrate that microsphere formulations of antimycobacterial drugs such as rifampin can be used for therapy of tuberculosis with minimal dosing.

Use of microsphere technology for targeted delivery of rifampin to Mycobacterium tuberculosis-infected macrophages.

Microsphere technology was used to develop formulations of rifampin for targeted delivery to host macrophages. These formulations were prepared by using biocompatible polymeric excipients of lactide and glycolide copolymers. Release characteristics were examined in vitro and also in two monocytic cell lines, the murine J774 and the human Mono Mac 6 cell lines. Bioassay assessment of cell culture supernatants from monocyte cell lines showed release of bioactive rifampin during a 7-day experimental period. Treatment of Mycobacterium tuberculosis H37Rv-infected monocyte cell lines with rifampin-loaded microspheres resulted in a significant decrease in numbers of CFU at 7 days following initial infection, even though only 8% of the microsphere-loaded rifampin was released. The levels of rifampin released from microsphere formulations within monocytes were more effective at reducing M. tuberculosis intracellular growth than equivalent doses of rifampin given as a free drug. These results demonstrate that rifampin-loaded microspheres can be formulated for effective sustained and targeted delivery to host macrophages.