Formulation and pharmacokinetics of self-assembled rifampicin nanoparticle systems for pulmonary delivery.

PURPOSE: To formulate rifampicin, an anti-tuberculosis antibiotic, for aerosol delivery in a dry powder 'porous nanoparticle-aggregate particle' (PNAP) form suited for shelf stability, effective dispersibility and extended release with local lung and systemic drug delivery. METHODS: Rifampicin was encapsulated in PLGA nanoparticles by a solvent evaporation process, spray dried into PNAPs containing varying amounts of nanoparticles, and characterized for physical and aerosol properties. Pharmacokinetic studies were performed with formulations delivered to guinea pigs by intratracheal insufflation and compared to oral and intravenous delivery of rifampicin. RESULTS: The PNAP formulations possessed properties suitable for efficient deposition in the lungs. In vitro release showed an initial burst of rifampicin, with the remainder available for release beyond eight hours. PNAPs delivered to guinea pigs by insufflation achieved systemic levels of rifampicin detected for six to eight hours. Moreover, rifampicin concentrations remained detectable in lung tissue and cells up to and beyond eight hours. Conversely, after pulmonary delivery of an aerosol without nanoparticles, rifampicin could not be detected in the lungs at eight hours. CONCLUSIONS: Our results indicate that rifampicin can be formulated into an aggregated nanoparticle form that, once delivered to animals, achieves systemic exposure and extends levels of drug in the lungs.

Pulmonary immunization of guinea pigs with diphtheria CRM-197 antigen as nanoparticle aggregate dry powders enhance local and systemic immune responses.

This study establishes the immune response elicited in guinea pigs after pulmonary and parenteral immunizations with diphtheria CRM-197 antigen (CrmAg). Several spray-dried powders of formalin-treated/untreated CrmAg nanoaggregates with L-leucine were delivered to the lungs of guinea pigs. A control group consisting of alum with adsorbed CrmAg in saline was administered by intramuscular injection. Animals received three doses of powder vaccines containing 20 or 40 µg of CrmAg. The serum IgG titers were measured for 16 weeks after the initial immunization; IgA titers were measured at the time of sacrifice in the broncho-alveolar lavage fluid. Further, toxin neutralization tests in naïve guinea pigs were performed for a few select serum samples. Histopathology of the lung tissues was conducted to evaluate inflammation or injury to the lung tissues. While the highest titer of serum IgG antibody was observed in guinea pigs immunized by the intramuscular route, those animals immunized with dry powder formulation by the pulmonary route, and without the adjuvant alum, exhibited high IgA titers. A pulmonary administered dry powder, compared to parenteral immunization, conferred complete protection in the toxin neutralization test. Mild inflammation was observed in lung tissues of animals receiving dry powder vaccines by the pulmonary route. Thus, administering novel CrmAg as dry powders to the lungs may be able to overcome some of the disadvantages observed with the existing diphtheria vaccine which is administered by the parenteral route. In addition, these powders will have the advantage of eliciting a high mucosal immune response in the lungs without using traditional adjuvants.

Pulmonary immunization using antigen 85-B polymeric microparticles to boost tuberculosis immunity.

This study aims to evaluate immunization with polymeric microparticles containing recombinant antigen 85B (rAg85B) delivered directly to the lungs to protect against tuberculosis. rAg85B was expressed in Escherichia coli and encapsulated in poly(lactic-co-glycolic acid) microparticles (P-rAg85B). These were delivered as dry powders to the lungs of guinea pigs in single or multiple doses of homologous and heterologous antigens. Bacille Calmette-Guérin (BCG) delivered subcutaneously was employed as the positive control and as part of immunization strategies. Immunized animals were challenged with a low-dose aerosol of Mycobacterium tuberculosis (MTB) H37Rv to assess the extent of protection measured as reduction in bacterial burden (CFU) in the lungs and spleens of guinea pigs. Histopathological examination and morphometric analysis of these tissues were also performed. The heterologous strategy of BCG prime-P-rAg85B aerosol boosts appeared to enhance protection from bacterial infection, as indicated by a reduction in CFU in both the lungs and spleens compared with untreated controls. Although the CFU data were not statistically different from the BCG and BCG-BCG groups, the histopathological and morphometric analyses indicated the positive effect of BCG-P-rAg85B in terms of differences in area of tissue affected and number and size of granulomas observed in tissues. P-rAg85B microparticles appeared to be effective in boosting a primary BCG immunization against MTB infection, as indicated by histopathology and morphometric analysis. These encouraging observations are relevant to boosting adults previously immunized with BCG or exposed to MTB, commonly the case in the developing world, and should be followed by further assessment of an appropriate immunization protocol for maximum protection.

Screening for potential adjuvants administered by the pulmonary route for tuberculosis vaccines.

Tuberculosis (TB) infects one third of the world's population, and new infections occur at a rate of 1/s. Better vaccines are needed than the live mycobacterium Bacille Calmette-Guérin (BCG). Alveolar macrophages (AMPhis) play a central role in pulmonary manifestations of TB. Targeting immunomodulators to AMPhis, the first line of defense against Mycobacterium tuberculosis (Mtb), may initiate a potent cell-mediated immune response. Muramyl dipeptide (MDP) and trehalose dibehenate (TDB) have elicited strong immune response when delivered to the lungs as aerosols. AMPhis show toxicity in response to some immunomodulators. The objective of this work was to screen the immunomodulators MDP and/or TDB encapsulated in microparticles (MPs) and to evaluate certain indicators of toxicity in human AMPhi-like cells. Poly(lactide-co-glycolide) (PLGA) MPs containing MDP and/or TDB were prepared by spray-drying. The morphology, particle size distribution, and immunomodulator encapsulation efficiency of MPs were examined. THP-1 cells were exposed to these MPs for 24 h and characteristics of cell morphology, tumor necrosis factor-alpha (TNF-alpha) release, lactate dehydrogenase (LDH), N-acetyl-beta-D: -glucosaminidase (NAG) and alkaline phosphatase (ALP) activity in AMPhi culture supernatants were measured. MTT assay was used to assess the viability of cells. Spray-drying produced low-density MPs having volume median diameters between 4 and 6 microm as measured by laser diffraction and projected area diameter between 3 and 5 microm calculated by microscopy. More TNF-alpha was produced by THP-1 cells exposed to MPs composed of PLGA-MDP or PLGA alone than PLGA-TDB. LDH release following exposure to MPs of PLGA-MDP and PLGA alone was greater than controls. NAG release was higher following exposure to MPs of PLGA alone or PLGA-MDP 0.1% than PLGA-TDB (0.1% and 1.0%). Cells remained viable after exposure to MPs as per MTT assay. PLGA-MDP MPs demonstrated statistically elevated indicators of biochemical responses in cell culture compared to PLGA-TDB MPs, but the extent of their potential to elicit adverse effects in vivo must be studied independently.

Poly (lactide-co-glycolide) microspheres in respirable sizes enhance an in vitro T cell response to recombinant Mycobacterium tuberculosis antigen 85B.

PURPOSE: To investigate the use of poly (lactide-co-glycolide) (PLGA) microparticles in respirable sizes as carriers for Antigen 85B (Ag85B), a secreted protein of Mycobacterium tuberculosis, with the ultimate goal of employing them in pulmonary delivery of tuberculosis vaccine. MATERIALS AND METHODS: Recombinant Ag85B was expressed from two Escherichia coli strains and encapsulated by spray-drying in PLGA microspheres with/without adjuvants. These microspheres containing rAg85B were assessed for their ability to deliver antigen to macrophages for subsequent processing and presentation to the specific CD4 T-hybridoma cells DB-1. DB-1 cells recognize the Ag85B(97-112) epitope presented in the context of MHC class II and secrete IL-2 as the cytokine marker. RESULTS: Microspheres suitable for aerosol delivery to the lungs (3.4-4.3 microm median diameter) and targeting alveolar macrophages were manufactured. THP-1 macrophage-like cells exposed with PLGA-rAg85B microspheres induced the DB-1 cells to produce IL-2 at a level that was two orders of magnitude larger than the response elicited by soluble rAg85B. This formulation demonstrated extended epitope presentation. CONCLUSIONS: PLGA microspheres in respirable sizes were effective in delivering rAg85B in an immunologically relevant manner to macrophages. These results are a foundation for further investigation into the potential use of PLGA particles for delivery of vaccines to prevent M. tuberculosis infection.