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 2.5×107 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.

Alginate nanoparticles as antituberculosis drug carriers: formulation development, pharmacokinetics and therapeutic potential.

BACKGROUND: Reduction in the dosing frequency of antituberculosis drugs (ATDs) by applying drug delivery technology has the potential to improve the patient compliance in tuberculosis (TB). Alginate (a natural polymer) based nanoparticulate delivery system was developed for frontline ATDs (rifampicin, isoniazid, pyrazinamide and ethambutol). METHODS: Alginate nanoparticles were prepared by the controlled cation induced gelification method and administered orally to mice. The drug levels were analysed by high performance liquid chromatography (HPLC) in plasma/tissues. The therapeutic efficacy was evaluated in M. tuberculosis H37Rv infected mice. RESULTS: High drug encapsulation efficiency was achieved in alginate nanoparticles, ranging from 70%-90%. A single oral dose resulted in therapeutic drug concentrations in the plasma for 7-11 days and in the organs (lungs, liver and spleen) for 15 days. In comparison to free drugs (which were cleared from plasma/organs within 12-24 h), there was a significant enhancement in the relative bioavailability of encapsulated drugs. In TB-infected mice three oral doses of the formulation spaced 15 days apart resulted in complete bacterial clearance from the organs, compared to 45 conventional doses of orally administered free drugs. CONCLUSIONS: Alginate nanoparticles appear to have the potential for intermittent therapy of TB.

Oral poly(lactide-co-glycolide) nanoparticle based antituberculosis drug delivery: toxicological and chemotherapeutic implications.

The present study reports on the detailed toxicological and chemotherapeutic evaluation of antituberculosis drug loaded nanoparticles in mice. A single oral dose administration of poly(lactide-co-glycolide) (PLG, a synthetic polymer) nanoparticles containing rifampicin+isoniazid+pyrazinamide+ethambutol could maintain drug levels in various tissues for 9-10 days and did not elicit any adverse response even when administered at several fold higher than the recommended therapeutic dose. However, dosing with conventional free drugs at the equivalent higher doses was lethal. Despite multiple oral dosing with the formulation at every 10th day, no toxicity was observed on the completion of subacute (28 days) or chronic (90 days) toxicity studies based on survival, gross pathology, histopathology, blood biochemistry and hematology. In mice harboring a high mycobacterial load (mimicking human tuberculosis), two independent chemotherapeutic regimens, i.e. 5 doses of PLG nanoparticles encapsulating (rifampicin+isoniazid+pyrazinamide+ethambutol) administered 10 days apart, or 2 doses of the 4-drug formulation followed by 3 doses of 2-drug formulation (rifampicin+isoniazid) resulted in undetectable bacilli. Further, the efficacy was comparable to 46 daily doses of oral free drugs. Therefore, the experimental evidence suggests that PLG nanoparticle-based antituberculosis drug delivery system is safe and well suited for prolonged and intermittent oral chemotherapy.

Nanotechnology based drug delivery system(s) for the management of tuberculosis.

The era of nanotechnology has allowed new research strategies to flourish in the field of drug delivery. Nanoparticle-based drug delivery systems are suitable for targeting chronic intracellular infections such as tuberculosis. Polymeric nanoparticles employing poly lactide-co-glycolide have shown promise as far as intermittent chemotherapy in experimental tuberculosis is concerned. It has distinct advantages over the more traditional drug carriers, i.e. liposomes and microparticles. Although the experience with natural carriers, e.g. solid lipid nanoparticles and alginate nanoparticles is in its infancy, future research may rely heavily on these carrier systems. Given the options for oral as well as parenteral therapy, the very nature of the disease and its complex treatment urges one to emphasize on the oral route for controlled drug delivery. Pending the discovery of more potent antitubercular drugs, nanotechnology-based intermittent chemotherapy provides a novel and sound platform for an onslaught against tuberculosis.

Liposomes as adjuvant for anti-mycobacterial vaccine development.

Mycobacteria are intracellular pathogens that invade and reside inside the macrophages. Recent advances in controlled delivery systems for vaccines such as liposomes have sparked a renewed interest in their potential application for the prevention of mycobacterial infections. The versatility of liposomes in the incorporation of hydrophilic/hydrophobic components, their non-toxic nature, biodegradability, biocompatibility, adjuvanticity, induction of cellular immunity, property of sustained release and prompt uptake by macrophages, makes them attractive candidates for the delivery of antigens. This review focuses on liposome research in the area of mycobacterial diseases and highlights how the various mycobacterial components may be exploited as powerful antigens with liposomes as adjuvants.

Lung specific stealth liposomes as antitubercular drug carriers in guinea pigs.

The problem of patient non-compliance in the management of tuberculosis (TB) can be overcome by reducing the dosing frequency of antitubercular drugs (ATD) employing drug carriers. This study reports on the intravenous (iv) administration of lung specific stealth liposomes encapsulating ATD (rifampicin and isoniazid in combination) to guinea pigs and the detailed pharmacokinetic/chemotherapeutic studies. Following a single iv administration of liposomal drugs, the latter were found to exhibit sustained therapeutic levels in plasma for 96-168 hr with half-lives of 24-70 hr, mean residence time (MRT) of 35-81 hr and organ drug levels up to day 7. The relative bioavailability (as compared to oral free drugs) was increased by 5.4-8.9 folds, whereas the absolute bioavailability (as compared to iv free drugs) was increased by 2.9-4.2 folds. Weekly therapy with liposomal ATD for 6 weeks produced equivalent clearance of Mycobacterium tuberculosis from organs as did daily therapy with oral free drugs. Hence, intravenous liposomal ATD offer the therapeutic advantage of reducing the dosing frequency and improving the patient compliance in the management of TB.

Recombinant mycobacterial proteins future directions to improve protective efficacy.

A large number of subunit vaccine candidates have recently been developed as alternatives to Mycobacterium bovis Bacillus Calmette-Guerin (BCG), which gives unpredictable and highly variable protection against tuberculosis. Immunological potential of various recombinant proteins against mycobacterial infections has been discussed. Further, strategies have been suggested, which include development of constructs coexpressing cytokines or regimens utilizing recombinant proteins for further improving the protective efficacy.

Evaluation of immune responses directed against 30kDa secretory protein of Mycobacterium tuberculosis H37Ra complexed in different adjuvants.

Ability of different adjuvants to promote cell mediated immune responses towards 30 kDa secretory protein of Mycobacterium tuberculosis H37Ra was monitored by assessing the lymphocyte proliferation and IgG1/IgG2a subclass profile in mouse model. Six formulations, viz. poly lactide-co-glycolide (PLG) microspheres, dimethyldioctadecyl ammoniumbromide (DDA), liposomes, liposomes containing monophosphoryl lipid A and coated with alum (L-LIPA-AL) or without alum (L-LIPA) were evaluated in comparison to standard Freund's incomplete adjuvant (FIA). Two adjuvant formulations of 30kDa-L-LIPA-AL and 30kDa-PLG showed maximum reactivity on VIIIth week post immunization (p.im) in terms of lymphoproliferation w.r.t. other adjuvant formulations. Both the vaccine formulations also exhibited a Th1 shift in terms of higher IgG2a response over IgGI. Flowcytometric analysis in the mesenteric lymph nodes (MLNs) of immunized animals revealed the capacity of 30kDa-PLG and 30kDa-L-LIPA-AL to activate T cell subsets like CD4 and CD8 T cells. The upregulation of B7 costimulatory molecules (B7-1 & B7-2) after immunization further proved the ability of the two vaccine formulations to activate antigen presenting cells. The immunostimulatory nature of the two formulations was also reflected in their capacity to reduce the bacilli load from the lungs of the experimentally infected mice. This study demonstrates PLG and L-LIPA-AL as potent adjuvants and their bioacceptibility and nontoxic nature make them suitable candidates for future subunit vaccine development against tuberculosis.

Biochemical effects of sparfloxacin on cell envelope of mycobacteria.

Sparfloxacin, a difluorinated quinolone is a potent anti-mycobacterial agent used in the treatment of mycobacterial infections. We have investigated whether sparfloxacin had other, more subtle effects on mycobacteria besides its interaction with DNA gyrase that could contribute to its therapeutic efficacy. Mycobacterium smegmatis cells grown in media with sub-inhibitory concentration of sparfloxacin were observed to have significant reduction in the biosynthesis of vital macromolecules, as shown by the incorporation of various radiolabelled precursors. The analysis of subcellular distribution of phospholipids of sparfloxacin-treated cells demonstrated an increase in the cell membrane and reduction in the cell wall, suggesting changes in the cell envelope architecture by sparfloxacin. Significant changes were also observed in other chemical constituents of the cell wall, especially in the arabinose and glucosamine contents. Mycolic acids, the major component of mycobacterial cell wall were reduced in the presence of MIC50 of sparfloxacin. There was a decrease in the limiting fluorescence intensity (Fmax) of 1-anilinonaphthalene 8-sulfonate (ANS) indicating alterations in the organization and conformation of mycobacterial cell surface. These results suggest that the mechanism of action of anti-mycobacterial action of sparfloxacin involves mycobacterial cell envelope.

Biochemical mechanism of combined effect of ethambutol and sparfloxacin against Mycobacterium smegmatis.

M. smegmatis cells grown in the presence of combination of ethambutol (EMB) and sparfloxacin (SPX) had decreased level of total cellular lipids as compared to control as well as cells grown in the presence of sub-inhibitory concentration (MIC50) of individual drugs. Amongst various phospholipids analyzed, maximum decrease was observed in the content of phosphatidylinositolmannosides (PIMs) of the cells grown in combination of EMB and SPX. In contrast, the subcellular distribution of phospholipids revealed a significant increase in PIMs content of both cell wall and cell membrane of the cells grown in the presence of combination of drugs as compared to control as well as individual drugs. Mycolic acids of M. smegmatis cells were found to be main targets as combination of drugs resulted in significant decrease in total cellular as well as cell wall mycolic acids as compared to control and individual drugs. Changed lipid composition of M. smegmatis cells grown in the presence of MIC50 of EMB, SPX and combination resulted in significant surface changes as was evident from decreased limiting fluorescence (Fmax) intensity of 1-anilinonaphthalene-8-sulfonate (ANS). Thus, the results of this study suggested that ethambutol and sparfloxacin in combination exerted their antimycobacterial effect principally due to their action on phosphatidylinositolmannosides (PIMs) and mycolic acids, which form the permeability barrier of mycobacteria.

Poly (DL-lactide-co-glycolide) microparticles as carriers for antimycobacterial drug rifampicin.

Poly (DL-lactide-co-glycolide) polymers were investigated as carriers for the first line antitubercular drug rifampicin. Different formulations of PLG microparticles viz. porous, non porous and hardened exhibited sustained release of rifampicin up to 7 weeks in vitro. However, hardened PLG microparticles exhibited the most sustained release in vivo in different organs up to 6 weeks. In case of free rifampicin, release was detected in vivo only up to 48 hr. In addition, no hepatotoxicity was observed on a biochemical basis (levels of SGPT, ALP and total bilirubin) in comparison to control animals. Taken together, these results suggest that polymer encapsulated antitubercular drug rifampicin may serve as an ideal therapeutic approach for treatment of tuberculous infections.

Poly (DL-lactide-co-glycolide) based delivery systems for vaccines and drugs.

Current vaccination and drug delivery strategies emphasize on the development of controlled release techniques for persistent and sustained effects. In the recent years, polymer based systems for the delivery of bioactive agents have gained considerable attention due to their marked adjuvanticity, established biodegradability and biocompatibility, excellent mechanical strength and controlled release profiles. This review deals with the potential applications of synthetic polymers mainly PLG polymers in delivery of vaccines and drugs.

Mycobacterial proteins--immune targets for antituberculous subunit vaccine.

Cellular and humoral immunity induced by Mycobacterium tuberculosis has led to identification of newer vaccine candidates, but despite this, many questions concerning the protection against tuberculosis remain unanswered. Recent progress in this field has centered on T cell subset responses and cytokines that these cells secrete. There has been a steady progress in identification and characterization of several classes of major mycobacterial proteins which includes secretory/export proteins, cell wall associated proteins, heat shock proteins and cytoplasmic proteins. The protein antigens are now believed to represent the key protective immunity inducing antigens in the bacillus. In this review, various mycobacterial protein antigens of vaccination potential are compared for their efficacy in light of current immunological knowledge.

Mechanism and specificity of immunoprotection induced by mycobacterial proteins against experimental tuberculosis in mice.

Mechanism of immunoprotection and specificity of two highly immunoprotective mycobacterial proteins, viz. 71 and 30 kDa were investigated. The adoptive transfer studies indicated that immunoprotection was mainly mediated by cooperative effect of CD4+ and CD8+ (66.7-73.3% on the basis of percent survival) which was further enhanced marginally by supplementation of B cells, natural killer cells, dendritic cells, macrophages and other immune cells. The specificity studies indicated that both the proteins did not cross react with the unrelated intracellular pathogens i.e. Aspergillus fumigatus, Salmonella typhi and Leishmania donovani as seen by T cell proliferation assay. The protection imparted by these mycobacterial proteins was also specific as the 71 and 30 kDa primed mice did not exhibit any cross protection against sublethal challenge of S typhi. The results indicate 71 and 30 kDa mycobacterial proteins to contain T cell specific epitopes responsible for specific immunoprotection, thus indicating their potential role as antituberculous vaccine candidates.

Immunoreactivity of mycobacterial 70 kDa protein in different physico-chemical forms.

The immunoreactivity of 70 kDa culture filtrate protein of Mycobacterium tuberculosis H37 Ra has been examined as such and by increasing its hydrophobicity through its conjugation to stearic acid ester (70 kDa-FAester). The cell mediated immune responses produced by 70 kDa-FAester encapsulated in phosphatidylcholine (PC) liposomes were significantly higher particularly at the 3rd week post immunization (wk p. im.) than that produced by the 70 kDa protein in PC liposomes, whereas humoral immune responses were non-significant. Further, these immune responses were comparable to that elicited by 70 kDa protein complexed with Freund's incomplete adjuvant (70 kDa-FIA). Results of this study suggest that changes in physicochemical nature of 70 kDa protein influences both the humoral and cellular immunoreactivity.

Immunoprophylactic studies on cell wall associated proteins of Mycobacterium tuberculosis H37Ra.

The cell wall protein peptidoglycan complex (CW-PPC) of Mycobacterium tuberculosis H37Ra was isolated through sequential extraction of lipids, carbohydrates and soluble proteins. CW-PPC emulsified in FIA was found to induce significant protection in mice against challenge with LD50 dose of M. tuberculosis H37Rv. To identify the immunoprotective components of CW-PPC, the proteins in avid association with peptidogican were dissociated by chemical treatment with trifluoromethanesulthonic acid (CF3CO3H): anisole (2:1). Immunoreactivity of total (CW-Pr) as well as its component proteins i.e., 71, 60 and 45 kDa proteins of cell wall was studied in animals immunized with CW-Pr-FIA. The 71 kDa protein was found to be most immunoreactive giving higher T-cell sensitization and humoral responses. Further, immunization of mice with 71 kDa-FIA demonstrated enhanced T- and B-cell responses. Mice immunized with 71 kDa-FIA gave significantly higher protection (P ≤ 0·05) against intravenous challenge with LD50 dose of M. tuberculosis H37Rv, than BCG immunized animals. The results indicate the potential of 71 kDa cell wall protein as a suitable candidate for Cthe subunit vaccine.

Changes in the cellular composition of Candida albicans resistant to miconazole.

Biochemical changes that accompany acquisition of miconazole resistance in a single step mutant of C. albicans 3153 were analyzed. Experiments show that resistance to this drug was associated with decrease in total lipids, phospholipids and sterol content. Fluorescence polarization studies with 1,6-diphenyl-1,3,5-hexatriene (DPH) showed decrease in polarization value (p) in resistant cells, thus indicating changes in membrane fluidity. Uptake of [3H] proline by intact cells revealed decrease in K(m) and Vmax of high affinity system (S1) of proline transport in cells resistant to miconazole. Results of this study suggest that membrane sensitivity of miconazole is determined by overall membrane organisation rather than by affinity of antifungal drug(s) for a single membrane component.

Calcium induced alterations in structural and functional role of phospholipids in Microsporum gypseum.

The effect of calcium on the structural and functional aspects of phospholipids in Microsporum gypseum was examined. Cells grown in presence of calcium exhibited increased content of phospholipids and enhanced synthesis of phospholipids as monitored by the incorporation of [32P] orthophosphoric acid. The rise in the levels of phospholipids was found to be due to increased synthesis of fatty acids as observed from [14C] acetate incorporation studies. The rise in the levels of phospholipids were reflected in the subcellular fractions also. Change in the phospholipid composition increased the fluidity of the membrane as is evident from fluorescence polarization studies using 1-anilinonaphthalene-8-sulfonate (ANS) and 1,6-diphenyl-1,3,5-hexatriene (DPH). The increased membrane fluidity was consistent with the enhanced uptake of [3H] proline in calcium grown cells.

Correlation between phospholipid biosynthesis and intracellular levels of cyclic adenosine monophosphate in Mycobacterium smegmatis.

The influence of intracellular levels of cAMP on phospholipid synthesis in Mycobacterium smegmatis has been examined under conditions of varying carbon source. A decreased phospholipid content was observed in glucose-grown cells, possibly due to decrease in intracellular cAMP levels caused by decreased/increased activity of adenylate cyclase and phosphodiesterase, respectively. The lowered phospholipid content was supported by decrease both in [14C]acetate incorporation and activities of key enzymes of phospholipid biosynthesis. These results in the light of our earlier observation of enhanced phospholipid synthesis in presence of increased levels of cAMP suggest a direct correlation between phospholipid biosynthesis and intracellular levels of cAMP in M. smegmatis.