Efficacy of moxifloxacin & econazole against multidrug resistant (MDR) Mycobacterium tuberculosis in murine model.

BACKGROUND & OBJECTIVES: Studies have shown the bactericidal potential of econazole and clotrimazole against Mycobacterium tuberculosis under in vitro and ex vivo conditions along with their synergism with conventional antituberculosis drugs. These molecules were also found to be effective against different multidrug resistant (MDR) M. tuberculosis isolates in vitro. Hence the present study was designed to evaluate the in vivo antimycobacterial potential of moxifloxacin and econazole alone and in combination against multidrug resistant tuberculosis (MDR-TB) in a mice model. METHODS: Mice were infected with 2.5×10 [7] bacilli of MDR strain of M. tuberculosis by aerosol route of infection. After four weeks of infection, chemotherapy was started orally by moxifloxacin 8.0 mg/kg body wt and econazole 3.3 mg/kg alone and in combination, as well as with four first line anti-tuberculosis drugs as a positive control. The animals were sacrificed and the lungs and spleen were excised under aspetic conditions. The tissues were homogenized with sterile normal saline, an aliquot of the homogenate was plated on Middlebrook 7H11 agar supplemented with oleate albumin dextrose catalase (OADC) and incubated at 37°C for four weeks. The number of visible and individual colonies were counted. RESULTS: The first line anti-tuberculosis drugs (RIF+INH+EMB+PZA) after eight weeks of therapy had no impact as the bacillary load in lungs and spleens remained unchanged. However, econazole, moxifloxacin alone as well as in combination significantly reduced the bacillary load in lungs as well as in spleens of MDR-TB bacilli infected mice. INTERPRETATION & CONCLUSIONS: Co-administration of the two drugs (econazole and moxifloxacin) to MDR-TB strain JAL-7782 infected mice exhibited additive effect, the efficacy of the drugs in combination being higher as compared with ECZ or MOX alone. These results were substantiated by histopathological studies. This study suggests the utility of econazole for the treatment of MDR tuberculosis and warrants further work in this direction.

Determination of glass transition temperature and in situ study of the plasticizing effect of water by inverse gas chromatography.

PURPOSE: To use an inverse gas chromatographic (IGC) method to determine the glass transition temperature (Tg) of some amorphous pharmaceuticals and to extend this technique for the in situ study of the plasticizing effect of water on these materials. METHODS: Amorphous sucrose and colyophilized sucrose-PVP mixtures were the model compounds. Both IGC and differential scanning calorimetry (DSC) were used to determine their Tg. By controlling the water vapor pressure in the IGC sample column, it was possible to determine the Tg of plasticized amorphous phases. Under identical temperatures and vapor pressures, the water uptake was independently quantified in an automated water sorption apparatus. RESULTS: The Tg of the dry phases, determined by IGC and by DSC, were in very good agreement. With an increase in the environmental relative humidity (RH), there was a progressive decrease in Tg as a result of the plasticizing effect of water. Because the water uptake was independently quantified, it was possible to use the Gordon-Taylor equation to predict the Tg values of the plasticized materials. The predicted values were in very good agreement with those determined experimentally using IGC. A unique advantage of this technique is that it provides complete control over the sample environment and is thus ideally suited for the characterization of highly reactive amorphous phases. CONCLUSIONS: An IGC method was used (a) to determine the glass transition temperature of amorphous pharmaceuticals and (b) to quantify the plasticizing effect of water on multicomponent systems.

Predicting solubility in multiple nonpolar drugs-cyclodextrin system.

This study presents a model to predict the solubility of a nonpolar drug D(A) in the presence of other nonpolar drugs D(1) em leader D(n) in a complexing ligand L system such as hydroxypropyl-beta-cyclodextrin (HPbetaCD). Using an equilibrium approach, the model describes the molecular interactions among these drug species and the ligand. The model indicates that the solubility of D(A) invariably decreases as a result of the presence of D(1) em leader D(n). Furthermore, the decrease in D(A) solubility is related to the sum of the products of the intrinsic solubilities of the other drugs and drug-ligand complexation constants. To test the model, three steroids (prednisolone, 17alpha-hydroxyprogesterone, and progesterone) were used as model compounds in HPbetaCD solutions. The experimental data showed that the solubility of any particular drug decreased in the presence of other drugs. At all tested HPbetaCD concentrations, these experimental solubility data were in good agreement with the predicted solubility data. This result lends strong support to the reliability and effectiveness of the proposed model.

Crystallization behavior of mannitol in frozen aqueous solutions.

PURPOSE: To study the effect of cooling rate, the influence of phosphate buffers and polyvinylpyrrolidone (PVP) on the crystallization behavior of mannitol in frozen aqueous solutions. METHODS: Low-temperature differential scanning calorimetry and powder X-ray diffractometry were used to characterize the frozen solutions. RESULTS: Rapid cooling (20 degrees C/min) inhibited mannitol crystallization, whereas at slower cooling rates (10 degrees C and 5 degrees C/min) partial crystallization was observed. The amorphous freeze-concentrate was characterized by two glass transitions at -32 degrees C and -25 degrees C. When the frozen solutions were heated past the two glass transition temperatures, the solute crystallized as mannitol hydrate. An increase in the concentration of PVP increased the induction time for the crystallization of mannitol hydrate. At concentrations of > or =100 mM, the buffer salts significantly inhibited mannitol crystallization. CONCLUSIONS: The crystallization behavior of mannitol in frozen solutions was influenced by the cooling rate and the presence of phosphate buffers and PVP.

The effect of antigen presenting cells on the cytokine profiles of stable and reactional lepromatous leprosy patients.

In view of varied reports on the Th1/Th2 paradigm in leprosy, we used a novel real time (RT) fluorogenic reverse transcriptase based PCR (RT-PCR) to measure cytokine expression in peripheral blood cells from lepromatous leprosy patients with stable disease and those suffering from erythema nodosum leprosum (ENL/Type II) reactions. To evaluate the role of accessory cells in Th cell differentiation, co-expression of Th cytokines interferon gamma (IFNgamma) and interleukin (IL) 4 and regulatory cytokines IL 10 and IL 12 was compared in antigen stimulated peripheral blood mononuclear cells (PBMC), cultures containing T cells reconstituted with autologous monocytes (MO) and cultures containing T cells reconstituted with autologous dendritic cells (DC). 7/8 stable lepromatous leprosy patients showed co-expression of both IFNgamma and IL 4, suggesting a Th0 or a combination of Th1 + Th2 subsets in PBMC. The RT-PCR demonstrated that stable lepromatous patients and patients in ENL had significantly higher levels of IFNgamma mRNA molecules compared to IL 4. In fact, 5/8 ENL patients had undetectable levels of IL 4 mRNA, with a skewing of the cytokine response towards a Th1-like profile. Consistent with this. IL 12p40 mRNA molecules were significantly higher in the PBMC of ENL patients compared to stable lepromatous patients (P < 0.01). Reconstitution of purified T cells with autologous DC and MO from the stable lepromatous group resulted in down regulation of IL 4 (P < 0.03 for DC and P < 0.02 for MO) and IL 10 (P < 0. 01 for DC and P < 0.02 for MO), and a consequent skewing towards a Th1 profile similar to that seen in ENL patients. The fact that accessory cells could alter the cytokine profile in the reconstituted cultures suggests that they may play a role in determining Th subset differentiation in chronic diseases, and may influence the immunological stability of such diseases.

Dysregulation of IL-4 expression in lepromatous leprosy patients with and without erythema nodosum leprosum.

In order to increase our understanding of the immunological basis of erythema nodosum leprosum (ENL), we studied Th-like cytokine profiles in 130 leprosy patients, employing both the conventional and a novel, real-time, fluorogenic reverse transcriptase-based PCR (RT-PCR). The concomitant expression of both Th-like cytokines, interferon-gamma and IL-4, and the regulatory cytokines, IL-10 and IL-12, was studied in the peripheral blood cells of leprosy patients with and without ENL. In the conventional RT-PCR, varied cytokine profiles were observed in individual patients of all clinical types. Fifty-three percent of lepromatous patients without ENL and 59% of tuberculoid leprosy patients showed co-expression of IFN gamma and IL-4, indicating a non-polarized Th 0 pattern. Of the 36 patients with ENL, 58% demonstrated a polarized Th 1 pattern, with only 30% expressing both cytokines. Semiquantitative RT-PCR indicated a lower expression of IL-4 compared to that of IFN gamma in the lepromatous patients without ENL; the difference was even greater among those with ENL. The sensitive, real-time PCR confirmed the down-regulation of IL-4 and IL-10, with absence of IL-4 in half of the patients, resulting in skewing of the cytokine response toward a Th 1-like profile.

Antisphingolipid antibodies in the sera of leprosy patients.

Earlier we reported the presence of significant levels of antigalactocerebroside (GalC) antibodies in the sera of leprosy patients. This study corroborates the above result and also gives evidence for the presence of antibodies to the nonpolar ceramide (Cer) moiety of GalC. AntiCer antibody titres were higher as compared to antiGalC antibodies in all categories of leprosy. The specificity of antibodies directed to the Cer moiety was confirmed using Lactosyl-BSA and neutralization assays. Statistically significant and positive correlations were observed between antiGalC and antiCer antibodies. Responsiveness factors were computed using natural logarithmic transformation of the variables.

Immunoreactivity of nerve lipid antigens in leprosy.

Neural lipid antigens, namely, galactocerebroside and ganglioside, have been implicated in demyelinating diseases. We were interested in finding the role of these antigens in leprosy neuritis. The humoral immune response to these lipid antigens was quantitated by enzyme-linked immunosorbent assay in sera from 91 leprosy patients and 18 normal individuals. Our data revealed the presence of antibodies to total nerve lipids (TNL) and galactocerebroside (GalC) and a significantly low level to ganglioside (Gg) in all the categories of leprosy. No antilipid antibodies were detected in normals. Anti-TNL and anti-GalC antibodies were highest in tuberculoid leprosy patients. Statistically significant positive correlation was observed between anti-TNL and anti-GalC antibodies in lepromatous borderline, tuberculoid, and neuritic patients.

Macrophage membrane alterations in leprosy as determined by change in sialic acid level.

The level of sialic acid removable by neuraminidase from macrophages of bacteriologically-positive lepromatous leprosy (B(+)LL) patient is extremely low, compared to macrophages from tuberculoid leprosy patients or normal individuals. On the other hand macrophages from long term treated bacteriologically-negative lepromatous leprosy (B(-)LL) patients show a much higher level of sialic acid. This higher level is drastically reduced when these macrophages from (B(-)LL) patients are allowed to phagocytose Mycobacterium leprae. This modulation could be host- and pathogen-specific. It is demonstrated that M. leprae infection brings out membrane changes in the macrophages leading to alteration in the surface molecules. Such membrane changes may cause hindrance in the ability of macrophages to participate successfully in the immune process.