Figure 7. Efficacy of D4476 in the murine model of Mtb infection.

Groups of naive mice (Female BALB/c mice 4–6 wk of age at 4/group) were infected with 1×10⁶ M. tuberculosis H37Rv via the tail vein. One group of mice was sacrificed 24 h later and lung homogenates were plated onto 7H11 agar plates for confirming infection. At ten days post infection, compound D4476 at either 2 nmol/g (1) or 4 nmol/g of body weight (2) was injected into the tail vein of mice for treatment. The i.v. injection was repeated on seventh day of treatment. In addition the inhibitor was also given intraperitoneally on the third, fifth, tenth and twelfth day after initiation of treatment. Here, a parallel group of mice received an identical treatment regimen with the vehicle only (Control). At fourteen days following treatment, mice were sacrificed and lung enriched using a homogenizer. An aliquot of the homogenate was lysed and plated onto 7H11 agar plates in serial dilutions for determining the mycobacterial load. The results are shown as mean (± SD) CFU values in panel A. This experiment was also repeated with the higher dose of D4476 (i.e. with 4 nmol/g of body weight with 4 mice/group). Here, the mean CFU values (± SD) obtained for the Control (vehicle only) and the D4476-treated groups were 68±1.4×10³ and 8.0±1.8×10³ respectively. Panel B shows the results of a similar experiment performed in mice that were infected with H37Rv through the aerosol route as described in Methods. Here 8 mice were used in each group and the values (mean ± S.D.) obtained in lung homogenates at 5 weeks after infection are shown. Here again the control group was that which received the vehicle only (i.e. DMSO) in parallel with D4476 administration in the other groups. Shown in Panel C are the mean (± S.D.) weight of the spleens obtained from either uninfected mice (Uninfected), infected mice without any inhibitor treatment (Control), or infected mice treated with either 2 (1) or 4 (2) nmol/g of body weight of D4476. These spleens are from the experiment described in Panel B. For panels A to C the p values for individual differences are indicated. Panel D shows lung sections of infected mice stained with hematoxylin / eosin 24 days after infection at ×10 (a) and ×40 (b). The typical pathology associated with experimental tuberculosis infection in mice is seen. Multiple well-formed epithelioid cell granulomas with mantle of lymphocytes are seen (indicated by the arrows). In contrast, mice treated with D4476 (160µg/kg body weight) maintained almost normal lung architecture ×10 (d), ×40 (e) with non-specific inflammation, and no granuloma formation was observed in the lungs of these mice. Further, untreated mice reveal large number of acid-fast bacilli when visualized at 100× (c) (indicated by the arrows); whereas very few bacilli were observed in mice treated with D4476 (4 nmol/g body weight) (f).