Bio-evaluation of poly(lactic-co-glycolic) acid nanoparticles loaded with radiolabelled rifampicin.

AIMS: The poly(lactic-co-glycolic) acid (PLGA) nanoparticles of tubercular drugs have been demonstrated to have a sustained release profile over 7 days. There is no information on the location or mode of release of these nanoparticles in living systems. Therefore, we have planned the study to explore the pharmacokinetics and biodistribution of PLGA rifampicin nanoparticles in healthy human volunteers. We aim to study the distribution pattern of PLGA-loaded nano-formulation of radiolabelled rifampicin in humans. METHODS: Rifampicin was labelled with 99m Tc by indirect method and nanoparticles were prepared by a single emulsion evaporation method. To investigate the pharmacokinetics and biodistribution of nanoparticles, a single dose of 450 mg of rifampicin was administered orally to healthy human volunteers divided into two different groups. RESULTS: Following a single oral dosage of the rifampicin nanoformulation, the pharmacokinetic (PK) parameters were significantly different between the nanoparticle and conventional groups: area under the concentration-time curve (AUC = 113.8 vs. 58.6; P < .001), mean residence time (MRT = 16.2 vs. 5.8; P < .01) and elimination rate constant (Ke = 0.04 vs. 0.10; P < .05). Also, Single-photon emission computed tomography/computed tomography (SPECT/CT) images revealed biodistribution of nanoparticles in the distal portions of the intestine, which is consistent with our dosimetry analysis. CONCLUSIONS: Significant difference in PK parameters and biodistribution of nanoparticles in spleen and lymph nodes with maximum deposition were observed in the large intestine. The nanoparticle distribution pattern may be advantageous for the treatment of intestinal or lymph node tuberculosis (TB) and has the potential to result in a lower dose of rifampicin nanoformulation for the treatment of pulmonary TB.

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.

Single ascending dose safety, tolerability, and pharmacokinetic study of econazole in healthy volunteers.

INTRODUCTION: Econazole has been found efficacious as antitubercular in in vitro and in vivo animal studies. However, limited information is available for its safety and pharmacokinetics in humans. In our present study we have conducted single ascending dose, safety, and pharmacokinetic evaluation in healthy human volunteers with the purpose of enabling translation for tuberculosis. METHODS: This study was conducted as a single-center, ascending-dose, placebo-controlled, double blind design. Three ascending dose were chosen (250 , 500 , and 1000 mg) to be administered as a single oral dose. The volunteers were screened for potential eligibility. Participants were randomized to receive either Econazole or Placebo in a 6:2 design. Safety assessments and pharmacokinetic evaluations were carried out for each cohort. RESULTS: Econazole was found to be safe at all dose levels. No serious or severe adverse events occurred during the study. The AUC (0-∞) showed a response relationship with a value of 49 ± 3.47 h* µg/ml, 17. 86 ± 8.40 hr* µg/ml, 35.54 ± 13.94 hr* µg/ml for 250 mg, 500 mg, and 1000 mg, respectively. CONCLUSION: Based on the findings of our study, a dose of 500 mg Econazole, once a day orally was considered as appropriate for further evaluation.

Novel chemotherapy for tuberculosis: chemotherapeutic potential of econazole- and moxifloxacin-loaded PLG nanoparticles.

The potential of econazole (ECZ) and moxifloxacin (MOX) individually against tuberculosis (TB) caused by multidrug-resistant and latent Mycobacterium tuberculosis has been demonstrated. In this study, poly-(dl-lactide-co-glycolide) (PLG) nanoparticle-encapsulated ECZ and MOX were evaluated against murine TB (drug susceptible) in order to develop a more potent regimen for TB. PLG nanoparticles were prepared by the multiple emulsion and solvent evaporation technique and were administered orally to mice. A single oral dose of PLG nanoparticles resulted in therapeutic drug concentrations in plasma for up to 5 days (ECZ) or 4 days (MOX), whilst in the organs (lungs, liver and spleen) it was up to 6 days. In comparison, free drugs were cleared from the same organs within 12-24h. In M. tuberculosis-infected mice, eight oral doses of the formulation administered weekly were found to be equipotent to 56 doses (MOX administered daily) or 112 doses (ECZ administered twice daily) of free drugs. Furthermore, the combination of MOX+ECZ proved to be significantly efficacious compared with individual drugs. Addition of rifampicin (RIF) to this combination resulted in total bacterial clearance from the organs of mice in 8 weeks. PLG nanoparticles appear to have the potential for intermittent therapy of TB, and combination of MOX, ECZ and RIF is the most potent.

Alginate-based sustained release drug delivery systems for tuberculosis.

Drug delivery systems have wide biomedical applications owing to their distinct therapeutic advantages, such as controlled release of drugs over prolonged periods, protection against premature drug degradation, reduction in drug toxicity and drug-drug interactions. All these factors are important considerations in the treatment of chronic infectious diseases such as tuberculosis. In tuberculosis, patient non-compliance is a vexing problem which is responsible not only for treatment failure, but also for the emergence of multi-drug resistant cases. Alginate, a natural polymer, has attracted researchers owing to its ease of availability, compatibility with hydrophobic as well as hydrophilic molecules, biodegradability under physiological conditions, lack of toxicity and the ability to confer sustained release potential. It is not therefore surprising that the controlled release phenomenon of this polymer has been documented for a vast array of drugs. In particular, the ability of alginate to co-encapsulate multiple antitubercular drugs and offer a controlled release profile is likely to have a major impact in enhancing patient compliance for better management of tuberculosis.

A multivalent combination of experimental antituberculosis DNA vaccines based on Ag85B and regions of difference antigens.

Two candidate DNA vaccines based on the proteins CFP10 and CFP21 encoded by regions of difference (RDs) of Mycobacterium tuberculosis were evaluated individually and in multivalent combination with the immunodominant protein Ag85B for induction of protective immune responses against experimental tuberculosis. Experimental DNA vaccines induced substantial levels of cell-mediated immune responses as indicated by marked lymphocyte proliferation, significant release of the Th1 cytokines IFN-gamma and IL-12 (p40), and predominant cytotoxic T cell activity. High levels of antigen-specific IgG1 and IgG2a antibodies observed in the sera of immunized mice depicted strong humoral responses generated by DNA vaccine constructs. The multivalent combination of three DNA vaccine constructs induced maximal T cell and humoral immune responses. All the experimental vaccines imparted significant protection against challenge with M. tuberculosis H(37)Rv (in terms of colony-forming unit reduction in lungs and spleen) as compared to vector controls. The level of protection exhibited by multivalent DNA vaccine formulation was found to be equivalent to that of Mycobacterium bovis BCG observed both at 4 and 8 weeks post-challenge. These results show the protective potential of the multivalent DNA vaccine formulation used in this study.

Azole antifungals as novel chemotherapeutic agents against murine tuberculosis.

The present study was designed to evaluate the in vivo antimycobacterial potential of econazole alone and in combination with antitubercular drugs against tuberculosis in mice. Econazole was found to reduce bacterial burden by 90% in the lungs and spleen of mice infected with 1 x 10(7) cells of Mycobacterium tuberculosis and was found to be equipotent to rifampicin. Further, our results indicate that econazole can replace rifampicin/isoniazid as well as both rifampicin and isoniazid in chemotherapy of murine tuberculosis. Econazole alone or in combination with antitubercular drugs did not produce any hepatotoxicity in normal or M. tuberculosis-infected mice.

Antimycobacterial activity of econazole against multidrug-resistant strains of Mycobacterium tuberculosis.

This study evaluated the antimycobacterial activity of econazole against multidrug-resistant (MDR) strains of Mycobacterium tuberculosis. The minimum inhibitory concentration (MIC(90)) and minimum bactericidal concentration (MBC(>99.99)) against MDR strains were found to be 0.120-0.125 microg/mL and 0.125-0.150 microg/mL, respectively, demonstrating the antimycobacterial potential of econazole.

Chemotherapeutic efficacy of nanoparticle encapsulated antitubercular drugs.

Our objective was to evaluate the chemotherapeutic potential of oral poly lactide-co-glycolide (PLG, a synthetic polymer) nanoparticle encapsulated ethambutol in combination with PLG-nanoparticle encapsulated-(rifampicin + isoniazid + pyrazinamide) in a murine tuberculosis (TB) model. Our formulation was prepared by the multiple emulsion technique and administered orally to mice for the biodistribution, pharmacokinetic, and chemotherapeutic studies. A single oral administration of the formulation to mice could maintain sustained drug levels in the plasma for 5 days and in the organs (lungs, liver, spleen) for 7-9 days. There was a striking improvement in the pharmacokinetic parameters such as half-life and mean residence time as compared with free drugs. The relative/absolute bioavailability of the 4 antituberculosis drugs was enhanced several fold. Repeated administration of the formulation did not produce any hepatotoxicity as assessed on a biochemical basis. In M. tuberculosis H37Rv infected mice, just 3 oral doses of the 4-drug formulation administered at every 10th day resulted in undetectable bacilli in the organs replacing 28 conventional doses of free drugs. We concluded that polymeric nanoparticle based oral 4-drug combination bears significant potential to shorten the duration of TB chemotherapy, besides reducing the dosing frequency.

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.

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.

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.

Effect of dibutyryl cyclic AMP on lipid synthesis in Microsporum gypseum.

The influence of intracellular levels of cAMP on the lipid synthesis of Microsporum gypseum has been examined by exogenous supplementation of dibutyryl cAMP and its activators/inhibitors. Incorporation of [14C]acetate into various lipid fractions of M. gypseum was markedly enhanced in the presence of dibutyryl cAMP and its modulators, probably as a consequence of increased intracellular cAMP levels, which, in turn, affected the lipid biosynthesis. Increased activities of phosphatidic acid phosphatase, glycerol kinase, ethanolamine kinase and choline kinase in the presence of these additives supports the enhanced synthesis of phospholipids and suggests that lipid biosynthesis is being controlled by cAMP in M. gypseum.

Influence of aminophylline on the lipids in Microsporum gypseum.

The effect of aminophylline on the lipid synthesis of Microsporum gypseum has been examined. A decreased incorporation of [14C]acetate into lipids was observed when the cells were incubated for 1 h with aminophylline which was reflected in all the individual lipid fractions. However, cells grown with aminophylline in the growth medium exhibited increased levels of total phospholipids, which was probably due to a rise in intracellular cAMP as these cells exhibited 4-fold increased levels of cAMP. Decreased activity of phosphodiesterase by aminophylline accounts for the increased cAMP levels. Increased phospholipid content in aminophylline grown cells was further supported by the increased incorporation of [14C]acetate into phospholipids as well as increased activities of phospholipid biosynthetic enzymes in comparison to non-supplemented cells.

Correlation between intracellular cAMP levels and phospholipids of Microsporum gypseum.

Atropine, a modulator of cAMP has been used to examine the relationship between phospholipids and intracellular levels of cAMP in Microsporum gypseum. A decreased phospholipid content was observed in atropine grown cells as a result of reduced levels of intracellular cAMP. This decline was caused by the inhibitory effect of atropine on adenylate cyclase. Lowered phospholipid content was supported by decreased [14C]acetate incorporation as well as reduced activities of key enzymes of phospholipid biosynthesis. In vitro supplementation of atropine in control cells also caused inhibition in lipid synthesis indicating similar effects of atropine and its metabolites. These results in conjunction with our previous report, in which enhanced levels of cAMP resulted in increased phospholipid synthesis, suggest a direct correlation between phospholipid biosynthesis and intracellular levels of cAMP in M. gypseum.

Lung specific stealth liposomes: stability, biodistribution and toxicity of liposomal antitubercular drugs in mice.

Liposomes with enhanced affinity towards lung tissue were prepared for the development of more effective chemotherapy against tuberculosis. Modification of surface of stealth liposomes by tagging O-stearylamylopectin (O-SAP) resulted in the increased affinity of these liposomes towards lung tissue of mice. Liposomes containing egg phosphatidylcholine (ePC), cholesterol (CH), dicetylphosphate (DCP), O-SAP and monosialogangliosides (GM1)/distearylphosphatidylethanolamine-poly(ethylene glycol) 2000 (DSPE-PEG 2000) were found to be most stable in serum. Tissue distribution of these liposomes showed more accumulation in lungs than in reticuloendothelial systems (RES) of normal and tuberculous mice. Pre administration of PC and CH (2:1.5) liposomes before the injection of lung specific stealth liposomes further enhanced their uptake in lungs. In vivo stability of these liposomes demonstrated the slow and controlled release of their encapsulated contents. Isoniazid and rifampicin encapsulated in liposomes were less toxic to peritoneal macrophages as compared to free drugs. Further, encapsulated drugs also demonstrated reduced in vivo toxicity in comparison to free drug(s). These findings suggest liposomes to be better drug delivery vehicles for experimental tuberculosis.

Cerulenin effect on phospholipid metabolism in Mycobacterium smegmatis ATCC 607.

Phospholipid metabolism in the presence of a subinhibitory concentration of cerulenin was studied in Mycobacterium smegmatis ATCC 607 by pulse labelling and subsequent chasing of radioactivity in phospholipids using [32P]orthophosphate. Cerulenin inhibited biosynthesis of total phospholipids to a significant level which is reflected equally in all the phospholipid components (phosphatidylethanolamine, phosphatidylinositol mannosides and cardiolipin) within the time of exposure. On chase, alteration in degradation of all phospholipid components was observed on cerulenin treatment, in comparison with control cells. Differences seen in the metabolism of phospholipids in cerulenin-treated and control cells are discussed.

Reversal of cerulenin-induced inhibition of phospholipids and sterol synthesis by exogenous fatty acids/sterols in Epidermophyton floccosum.

Cerulenin, a specific inhibitor of fatty acids and sterol biosynthesis inhibited the growth of Epidermophyton floccosum, which was reversed when growth medium was supplemented with palmitic acid and sterols. Unsaturated fatty acids partially restored the growth. Cerulenin inhibited both phospholipid and sterol biosynthesis (60-70%) at the minimum inhibitory concentration (0.5 microgram/ml) as demonstrated by [32P]orthophosphoric acid and [14C]acetate incorporation into the respective lipids. Cerulenin-induced inhibition of phospholipid and sterol synthesis was dose dependent up to 0.5 microgram/ml. Exogenously supplied fatty acids and sterols restored the biosynthesis of phospholipids in cerulenin-treated cultures, while that of sterols was enhanced. The biosynthesis of both saturated and unsaturated fatty acids was inhibited by cerulenin.

Purification and characterization of cAMP dependent protein kinase from Microsporum gypseum.

A cyclic AMP dependent protein kinase (PKA), its regulatory (R) and catalytic (C) subunits were purified to homogeneity from soluble extract of Microsporum gypseum. Purified enzyme showed a final specific activity of 277.9 nmol phosphate transferred min(-1) mg protein(-1) with kemptide as substrate. The enzyme preparation showed two bands with molecular masses of 76 kDa and 45 kDa on sodium dodecyl polyacrylamide gel electrophoresis. The 76 kDa subunit was found to be the regulatory (R) subunit of PKA holoenzyme as determined by its immunoreactivity and the isoelectric point of this subunit was 3.98. The 45 kDa subunit was found to be the catalytic (C) subunit by its immunoreactivity and phosphotransferase activity. Gel filtration using Sepharose CL-6B revealed the molecular mass of PKA holoenzyme to be 240 kDa, compatible with its tetrameric structure, consisting of two regulatory subunits (76 kDa) and two catalytic subunits (45 kDa). The specificity of enzyme towards protein acceptors in decreasing order of phosphorylation was found to be kemptide, casein, syntide and histone IIs. Purified enzyme had apparent K(m) values of 71 microM and 25 microM for ATP and kemptide, respectively. Phosphorylation was strongly inhibited by mammalian PKA inhibitor (PKI) but not by inhibitors of other protein kinases. The PKA showed maximum activity at pH 7.0 and enzyme activity was inhibited in the presence of N-ethylmaleimide (NEM) which shows the involvement of sulfhydryl groups for the activity of PKA. PKA phosphorylated a number of endogenous proteins suggesting the multifunctional role of cAMP dependent protein kinase in M. gypseum. Further work is under progress to identify the natural substrates of this enzyme through which it may regulate the enzymes involved in phospholipid metabolism.

Identification, localization and possible role of calmodulin like protein in phospholipid synthesis of Microsporum gypseum.

Calmodulin like protein has been identified for the first time in dermatophyte--M. gypseum (by specific radioimmunoassay). Maximum amount of this protein was present in the early and mid log phase of growth and was mainly localized in the cytosolic fraction. Cells treated or grown with calmodulin antagonists (phenothiazine and trifluoperazine) exhibited lower uptake of [14C]acetate or labelled phosphate into phospholipids. This is probably due to lower levels of calmodulin seen in these cells. Our results suggest the relationship between calmodulin levels and phospholipid synthesis in Microsporum gypseum.