Evaluation of anti-AIDS drugs in conventional mice implanted with a permeable membrane device containing human T cells infected with HIV.

We now report the confirmation of the work of Hollingshead et al. (1995) on development of a cell based hollow fiber (HF) system for evaluating potential anti-AIDS drugs in vivo using conventional mice rather than SCID mice. CD4 +, CEM-SS cells infected with HIV/1, strain RF, at a multiplicity of infection of 0.1 were placed into HFs. The fibers were implanted into the peritoneal cavity of outbred Swiss mice. Using this model, the antiviral activity of azidothymidine (AZT) at doses of approximately 150, 75 and 37.5 mg/kg/day was evaluated by administering AZT to the mice in drinking water. Upon fiber removal on day 6, AZT treatment was shown to significantly increase CEM cell viability over the untreated, virus control group and significantly reduced the levels of HIV p24 and HIV RT activity.

In vitro and in vivo enhancement of ddI activity against Rauscher murine leukemia virus by ribavirin.

Ribavirin has been reported to enhance the activity of ddI against HIV. We explored this enhancement of antiviral activity in Rauscher murine leukemia virus (RMuLV) models in vitro and in vivo. The significant finding in these studies was that combinations of the drugs exhibited virus titer reductions that were greater than would be expected if the drug interactions were simply additive. These effects were designated synergistic by the method of Prichard and Shipman (Prichard, M.N. and Shipman, C., Jr. (1990). A three-dimensional model to analyze drug-drug interaction, Antiviral Res. 14, 181-206). In addition to the antiviral synergy, we also observed some synergistic toxicity in the animal model.

Use of Mono Mac 6 human monocytic cell line and J774 murine macrophage cell line in parallel antimycobacterial drug studies.

The Mono Mac 6 (MM6) human monocytic cell line was evaluated with the established J774 murine macrophage cell line to ascertain its effectiveness in determining the intracellular activities of antimycobacterial drugs. Cells were infected with Mycobacterium tuberculosis H37Ra and treated with drug concentrations corresponding to the MICs, as well as to threefold higher than and threefold less than the MICs. Changes in CFU were compared after 7 days to determine significant differences between treated and nontreated groups. The results suggest that MM6 will make a useful model for testing the intracellular activities of antituberculosis drugs.

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.

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.

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.