Improved Mycobacterium tuberculosis clearance after the restoration of IFN-γ+ TNF-α+ CD4+ T cells: Impact of PD-1 inhibition in active tuberculosis patients.

Prolonged therapy, drug toxicity, noncompliance, immune suppression, and alarming emergence of drug resistance necessitate the search for therapeutic vaccine strategies for tuberculosis (TB). Such strategies ought to elicit not only IFN-γ, but polyfunctional response including TNF-α, which is essential for protective granuloma formation. Here, we investigated the impact of PD-1 inhibition in facilitating protective polyfunctional T cells (PFTs), bacillary clearance, and disease resolution. We have observed PD-1 inhibition preferentially rescued the suppressed PFTs in active tuberculosis patients. In addition, polyfunctional cytokine milieu favored apoptosis of infected MDMs over necrosis with markedly reduced bacillary growth (≪CFU) in our in vitro monocyte-derived macrophages (MDMs) infection model. Furthermore, the animal study revealed a significant decline in the bacterial burden in the lungs and spleen of infected mice after in vivo administration of α-PD-1 along with antitubercular treatment. Our findings suggest that rescuing polyfunctional immune response by PD-1 inhibition works synergistically with antituberculosis chemotherapy to confer improved control over bacillary growth and dissemination. In summary, our data strongly indicate the therapeutic potential of α-PD-1 as adjunct immunotherapy that can rejuvenate suppressed host immunity and enhance the efficacy of candidate therapeutic vaccine(s).

Activating transcription factor 3 modulates the macrophage immune response to Mycobacterium tuberculosis infection via reciprocal regulation of inflammatory genes and lipid body formation.

Infection of macrophages by Mycobacterium tuberculosis elicits an immune response that clears the bacterium. However, the bacterium is able to subvert the innate immune response. Differential expression of transcription factors (TFs) is central to the dynamic balance of this interaction. Among other functions, TFs regulate the production of antibacterial agents such as nitric oxide, pro-inflammatory cytokines and neutral lipids which are stored in lipid bodies (LBs) and favour bacterial survival. Here, we demonstrate that the TF activating transcription factor 3 (ATF3) is upregulated early during infection of macrophages or mice. Depletion of ATF3 enhances mycobacterial survival in macrophages suggesting its host-protective role. ATF3 interacts with chromatin remodelling protein brahma-related gene 1 and both associate with the promoters of interleukin-12p40, interleukin-6 and nitric oxide synthase 2, to activate expression of these genes. Strikingly, ATF3 downregulates LB formation by associating at the promoters of positive regulators of LB formation such as cholesterol 25 hydroxylase and the microRNA-33 locus. ATF3 represses the association of the activating mark, acetyl histone H4 lysine 8 at the promoter of cholesterol 25 hydroxylase. Our study suggests opposing roles of ATF3 in regulation of distinct sets of macrophage genes during infection, converging on a host-protective immune response.

The sensor kinase MtrB of Mycobacterium tuberculosis regulates hypoxic survival and establishment of infection.

Paired two-component systems (TCSs), having a sensor kinase (SK) and a cognate response regulator (RR), enable the human pathogen Mycobacterium tuberculosis to respond to the external environment and to persist within its host. Here, we inactivated the SK gene of the TCS MtrAB, mtrB, generating the strain ΔmtrB We show that mtrB loss reduces the bacterium's ability to survive in macrophages and increases its association with autophagosomes and autolysosomes. Notably, the ΔmtrB strain was markedly defective in establishing lung infection in mice, with no detectable lung pathology following aerosol challenge. ΔmtrB was less able to withstand hypoxic and acid stresses and to form biofilms and had decreased viability under hypoxia. Transcriptional profiling of ΔmtrB by gene microarray analysis, validated by quantitative RT-PCR, indicated down-regulation of the hypoxia-associated dosR regulon, as well as genes associated with other pathways linked to adaptation of M. tuberculosis to the host environment. Using in vitro biochemical assays, we demonstrate that MtrB interacts with DosR (a noncognate RR) in a phosphorylation-independent manner. Electrophoretic mobility shift assays revealed that MtrB enhances the binding of DosR to the hspX promoter, suggesting an unexpected role of MtrB in DosR-regulated gene expression in M. tuberculosis Taken together, these findings indicate that MtrB functions as a regulator of DosR-dependent gene expression and in the adaptation of M. tuberculosis to hypoxia and the host environment. We propose that MtrB may be exploited as a chemotherapeutic target against tuberculosis.

MicroRNA 26a (miR-26a)/KLF4 and CREB-C/EBPß regulate innate immune signaling, the polarization of macrophages and the trafficking of Mycobacterium tuberculosis to lysosomes during infection.

For efficient clearance of Mycobacterium tuberculosis (Mtb), macrophages tilt towards M1 polarization leading to the activation of transcription factors associated with the production of antibacterial effector molecules such as nitric oxide (NO) and proinflammatory cytokines such as interleukin 1 ß (IL-1ß) and tumor necrosis factor α (TNF-α). At the same time, resolution of inflammation is associated with M2 polarization with increased production of arginase and cytokines such as IL-10. The transcriptional and post-transcriptional mechanisms that govern the balance between M1 and M2 polarization, and bacteria-containing processes such as autophagy and trafficking of Mtb to lysosomes, are incompletely understood. Here we report for the first time, that the transcription factor KLF4 is targeted by microRNA-26a (miR-26a). During Mtb infection, downregulation of miR-26a (observed both ex vivo and in vivo) facilitates upregulation of KLF4 which in turn favors increased arginase and decreased iNOS activity. We further demonstrate that KLF4 prevents trafficking of Mtb to lysosomes. The CREB-C/EBPß signaling axis also favors M2 polarization. Downregulation of miR-26a and upregulation of C/ebpbeta were observed both in infected macrophages as well as in infected mice. Knockdown of C/ebpbeta repressed the expression of selected M2 markers such as Il10 and Irf4 in infected macrophages. The importance of these pathways is substantiated by observations that expression of miR-26a mimic or knockdown of Klf4 or Creb or C/ebpbeta, attenuated the survival of Mtb in macrophages. Taken together, our results attribute crucial roles for the miR-26a/KLF4 and CREB-C/EBPßsignaling pathways in regulating the survival of Mtb in macrophages. These studies expand our understanding of how Mtb hijacks host signaling pathways to survive in macrophages, and open up new exploratory avenues for host-targeted interventions.

Inhalable liposomes of Glycyrrhiza glabra extract for use in tuberculosis: formulation, in vitro characterization, in vivo lung deposition, and in vivo pharmacodynamic studies.

OBJECTIVE: The current study involves the development of liposomal dry powder for inhalation (LDPI) containing licorice extract (LE) for use in tuberculosis. SIGNIFICANCE: The current epidemiology of tuberculosis along with the increasing emergence of resistant forms of tuberculosis necessitates the need for developing alternative efficacious medicines for treatment. Licorice is a medicinal herb with reported activity against Mycobacterium tuberculosis. METHODS: Liposomes with LE were prepared by thin film hydration technique and freeze dried to obtain LDPI. The comprehensive in vitro and in vivo characterization of the LDPI formulation was carried out. RESULTS: The particle size of liposomes was around 210 nm with drug entrapment of almost 75%. Transmission electron microscopy revealed spherical shape of liposome vesicles. The flow properties of the LDPI were within acceptable limits. Anderson Cascade Impactor studies showed the mean median aerodynamic diameter, geometric standard deviation and fine particle fraction of the LDPI to be 4.29 µm, 1.23, and 54.68%, respectively. In vivo lung deposition studies of LDPI in mice showed that almost 46% of the drug administered reaches the lungs and 16% of administered drug is retained in the lungs after 24 hours of administration. The in vivo pharmacodynamic evaluation of the LDPI showed significant reduction in bacterial counts in lungs as well as spleen of TB-infected mice. CONCLUSIONS: LE LDPI thus has a promising potential to be explored as an effective anti-tubercular medicine or as an adjunct to existing anti-tubercular drugs.

MicroRNA 17-5p regulates autophagy in Mycobacterium tuberculosis-infected macrophages by targeting Mcl-1 and STAT3.

Autophagy plays a crucial role in the control of bacterial burden during Mycobacterium tuberculosis infection. MicroRNAs (miRNAs) are small non-coding RNAs that regulate immune signalling and inflammation in response to challenge by pathogens. Appreciating the potential of host-directed therapies designed to control autophagy during mycobacterial infection, we focused on the role of miRNAs in regulating M. tuberculosis-induced autophagy in macrophages. Here, we demonstrate that M. tuberculosis infection leads to downregulation of miR-17 and concomitant upregulation of its targets Mcl-1 and STAT3, a transcriptional activator of Mcl-1. Forced expression of miR-17 reduces expression of Mcl-1 and STAT3 and also the interaction between Mcl-1 and Beclin-1. This is directly linked to enhanced autophagy, because Mcl-1 overexpression attenuates the effects of miR-17. At the same time, transfection with a kinase-inactive mutant of protein kinase C δ (PKCδ) (an activator of STAT3) augments M. tuberculosis-induced autophagy, and miR-17 overexpression diminishes phosphorylation of PKCδ, suggesting that an miR-17/PKC δ/STAT3 axis regulates autophagy during M. tuberculosis infection.

Protective effect of antigen excess immune complex in guinea pigs infected with Mycobacterium tuberculosis.

Background & objectives: : Immune complexes (ICs) play a crucial role which can either be beneficial or pathological to the host. Involvement of circulating immune complexes (CICs) has been shown in tuberculosis (TB) cases (adults and neonates form), but its immunomodulatory effect has not been studied in vivo. Hence, this study was carried out to understand and explore the prognostic therapeutic potential of CICs on the host immune system in guinea pigs animal TB model. Methods: In this study, the guinea pigs (group I) were immunized with in vitro synthesized antigen excess IC (AgX-IC), group II with antibody excess IC (AbX-IC) and group III with phosphate-buffered saline. All these animals were sensitized with Mycobacterium tuberculosis H37Rv before immunization and subsequently infected with M. tuberculosis H37Rv strain post-immunization with IC. Results: Mortality was observed in animals belonging of groups II and III, while all animals in group I survived. A steady increase in the body weight of animals immunized with AgX-IC was observed when compared to the other groups. The infection load in the spleen and lungs was less in animals from group I when compared to the other groups. The CICs were found to be in higher concentration in serum of IC-immunized guinea pigs when compared to ICs non-immunized animals. Interpretation & conclusions: Based on our findings, it can be speculated that the ICs may have a protective immunomodulatory role pertaining to disease progression and development of pathology. As a new perspective, with further insight into the underlying mechanism of action and correlation with clinical data, ICs may also be used as a potential tool for assessing the immune status of the infected individuals, especially the close contacts of TB patients.

Modelling of cerebral tuberculosis in BALB/c mice using clinical strain from patients with CNS tuberculosis infection.

BACKGROUND & OBJECTIVES: Central nervous system (CNS) infection caused by Mycobacterium tuberculosis (MTB) is the most severe form of extrapulmonary tuberculosis (EPTB) due to a high level of mortality and morbidity. Limited studies are available on CNS-TB animal model development. The present study describes the development of a murine model of CNS-TB using a clinical strain (C3) isolated from the cerebrospinal fluid (CSF) of CNS-TB patients. METHODS: Groups of mice were infected by the intravenous route with MTB C3 strain isolated from the CSF of CNS-TB patients. Brain and lung tissue were evaluated for bacterial burden, histopathology and surrogate markers of TB infection at 30 and 50 days post-infection. RESULTS: Mice infected intravenously with MTB C3 strains showed progressive development of CNS disease with high bacillary burden in lungs at the initial stage (30 days), which eventually disseminated to the brain at a later stage (50 days). Similarly, high mortality (60%) was associated in mice infected with C3 strain compared to control. INTERPRETATION & CONCLUSIONS: The study showed development of a novel murine model of CNS-TB using the C3 strain of MTB that replicated events of extrapulmonary dissemination. The developed model would be helpful in understanding the pathogenesis of CNS-TB infection for the development of improved therapeutic interventions in future.

Inhalable Particles for "Pincer Therapeutics" Targeting Nitazoxanide as Bactericidal and Host-Directed Agent to Macrophages in a Mouse Model of Tuberculosis.

Nitazoxanide (NTZ) has moderate mycobactericidal activity and is also an inducer of autophagy in mammalian cells. High-payload (40-50% w/w) inhalable particles containing NTZ alone or in combination with antituberculosis (TB) agents isoniazid (INH) and rifabutin (RFB) were prepared with high incorporation efficiency of 92%. In vitro drug release was corrected for drug degradation during the course of study and revealed first-order controlled release. Particles were efficiently taken up in vitro by macrophages and maintained intracellular drug concentrations at one order of magnitude higher than NTZ in solution for 6 h. Dose-dependent killing of Mtb and restoration of lung and spleen architecture were observed in experimentally infected mice treated with inhalations containing NTZ. Adjunct NTZ with INH and RFB cleared culturable bacteria from the lung and spleen and markedly healed tissue architecture. NTZ can be used in combination with INH-RFB to kill the pathogen and heal the host.

Preparation and Preclinical Evaluation of Inhalable Particles Containing Rapamycin and Anti-Tuberculosis Agents for Induction of Autophagy.

PURPOSE: Mycobacterium tuberculosis (Mtb) inhibits host defense mechanisms, including autophagy. We investigated particles containing rapamycin (RAP) alone or in combination with isoniazid (INH) and rifabutin (RFB) for: targeting lung macrophages on inhalation; inducing autophagy; and killing macrophage-resident Mtb and/or augmenting anti-tuberculosis (TB) drugs. METHODS: PLGA and drugs were spray-dried. Pharmacokinetics, partial biodistribution (LC-MS/MS) and efficacy (colony forming units, qPCR, acid fast staining, histopathology) in mice following dry powder inhalation were evaluated. RESULTS: Aerodynamic diameters of formulations were 0.7-4.7 µm. Inhaled particles reached deep lungs and were phagocytosed by alveolar macrophages, yielding AUC0-48 of 102 compared to 0.1 µg/ml × h obtained with equivalent intravenous dose. RAP particles induced more autophagy in Mtb-infected macrophages than solutions. Inhaled particles containing RAP alone in daily, alternate-day and weekly dosing regimens reduced bacterial burden in lungs and spleens, inducing autophagy and phagosome-lysosome fusion. Inhalation of particles containing RAP with INH and RFB cleared the lungs and spleens of culturable bacteria. CONCLUSIONS: Targeting a potent autophagy-inducing agent to airway and lung macrophages alone is feasible, but not sufficient to eliminate Mtb. Combination of macrophage-targeted inhaled RAP with classical anti-TB drugs contributes to restoring tissue architecture and killing Mtb.

Development and Characterization of Nanoembedded Microparticles for Pulmonary Delivery of Antitubercular Drugs against Experimental Tuberculosis.

The foremost objective of the present research study was to develop and evaluate the potential of rifampicin (RIF) and isoniazid (INH) loaded spray dried nanoembedded microparticles against experimental tuberculosis (TB). In this study, RIF-INH loaded various formulations (chitosan, guar gum, mannan, and guar gum coated chitosan) were prepared by spray drying and characterized on the basis of in vitro as well as in vivo studies. Results showed that guar gum spray dried particles showed uniform size distribution with smooth surface as compare to mannan formulations. Guar gum batches exhibited excellent flow ability attributed to their optimum moisture content and uniform size distribution. The drug release showed the biphasic pattern of release, i.e., initial burst followed by a sustained release pattern. The preferential uptake of guar gum coated formulations suggested the presence and selective uptake capability of mannose moiety to the specific cell surface of macrophages. In vivo lung distribution study showed that guar gum coated chitosan (GCNP) batches demonstrated prolonged residence at the target site and thereby improve the therapeutic utility of drug with a significant reduction in systemic toxicity. Optimized drug loaded GCNP formulation has resulted in almost 5-fold reduction of the number of bacilli as compared to control group. Histopathology study also demonstrated that none of the treated groups show any evidence of lung tissue abnormality. Hence, GCNPs could be a promising carrier for selective delivery of antitubercular drugs to alveolar macrophages with the interception of minimal side effects, for efficient management of TB.

Constitutive expression of SMAR1 confers susceptibility to Mycobacterium tuberculosis infection in a transgenic mouse model.

BACKGROUND & OBJECTIVES: Studies involving animal models of experimental tuberculosis have elucidated the predominant role of cytokines secreted by T cells and macrophages to be an essential component of the immune response against Mycobacterium tuberculosis infection. The immune activities of CD4+ T cells are mediated in part by Th1 cytokine interferon gamma (IFN-γ) which is produced primarily by T cells and natural killer (NK) cells and critical for initiating the immune response against intracellular pathogen such as M. tuberculosis. Nuclear matrix protein SMAR1 plays an important role in V(D)J recombination, T helper cell differentiation and inflammatory diseases. In this study a transgenic mouse model was used to study the role of SMAR1 in M. tuberculosis infection. METHODS: Wild type BALB/c, C57BL/6, BALB/c-EGFP-SMAR1 and C57BL/6-SMAR1 transgenic mice were infected with M. tuberculosis (H37Rv). A dose of 100 bacilli was used for infection via respiratory route. Bacterial load in lung and spleen of infected mice was determined at 2, 4, 6 and 8 wk post-infection. Gene expression analysis for Th1 cytokines and inducible nitric oxide synthase (iNOS) was performed in infected lung tissues by quantitative reverse transcription (RT)-PCR. RESULTS: SMAR1 transgenic mice from both BALB/c and C57BL/6 genetic background displayed higher bacillary load and susceptibility to M. tuberculosis infection compared to wild type mice. This susceptibility was attributed due to compromised of Th1 response exhibited by transgenic mice. INTERPRETATION & CONCLUSIONS: SMAR1 transgenic mice exhibited susceptibility to M. tuberculosis infection in vivo irrespective of genetic background. This susceptibility was attributed to downregulation of Th1 response and its hallmark cytokine IFN-γ. Hence, SMAR1 plays an important role in modulating host immune response after M. tuberculosis infection.

Inhaled microparticles containing clofazimine are efficacious in treatment of experimental tuberculosis in mice.

Inhalable clofazimine-containing dry powder microparticles (CFM-DPI) and native clofazimine (CFM) were evaluated for activity against Mycobacterium tuberculosis in human monocyte-derived macrophage cultures and in mice infected with a low-dose aerosol. Both formulations resulted in 99% killing at 2.5 µg/ml in vitro. In mice, 480 µg and 720 µg CFM-DPI inhaled twice per week over 4 weeks reduced numbers of CFU in the lung by as much as log(10) 2.6; 500 µg oral CFM achieved a log(10) 0.7 reduction.

Promiscuous peptide of 16 kDa antigen linked to Pam2Cys protects against Mycobacterium tuberculosis by evoking enduring memory T-cell response.

One of the main reasons considered for BCG failure in tuberculosis-endemic areas is impediment by environmental mycobacteria in its processing and generation of memory T-cell response. To overcome this problem, we developed a unique lipopeptide (L91) by linking the promiscuous peptide (sequence 91-110) of 16 kDa antigen of Mycobacterium tuberculosis to Pam2Cys. L91 does not require extensive antigen processing and generates enduring Th1 memory response. This is evidenced by the fact that L91 significantly improved the activation, proliferation, and generation of protective T cells. Furthermore, L91 surmounts the barrier of major histocompatibility complex polymorphism and induces better protection than BCG. This peptide has self-adjuvanting properties and activates dendritic cells. Importantly, L91 activates T cells isolated from purified protein derivative-positive healthy volunteers that responded weakly to free peptide (F91). In essence, L91 can be a potent future vaccine candidate against tuberculosis.

Mycobacterium indicus pranii as stand-alone or adjunct immunotherapeutic in treatment of experimental animal tuberculosis.

BACKGROUND & OBJECTIVES: Mycobacterium w (M.w) is a saprophytic cultivable mycobacterium and shares several antigens with M. tuberculosis. It has shown good immunomodulation in leprosy patients. Hence in the present study, the efficacy of M.w immunotherapy, alone or in combination with multi drug chemotherapeutic regimens was investigated against drug sensitive M. tuberculosis H37Rv and three clinical isolates with variable degree of drug resistance in mice. METHODS: BALB/c mice were infected with M. tuberculosis H37Rv (susceptible to all first and second line drugs) and three clinical isolates taken from the epository of the Institute. The dose of 200 bacilli was used for infection via respiratory route in an aerosol chamber. Chemotherapy (5 days/wk) was given one month after infection and the vaccinated group was given a dose of 1x107 bacilli by subcutaneous route. Bacterial load was measured at 4 and 6 wk after initiation of chemotherapy. RESULTS: M.w when given along with chemotherapy (4 and 6 wk) led to a greater reduction in the bacterial load in lungs and other organs of TB infected animals compared to. However, the reduction was significantly (P<0.05) more in terms of colony forming units (cfu) in both organs (lungs and spleen). CONCLUSION: M.w (as immunomodulator) has beneficial therapeutic effect as an adjunct to chemotherapy.

Coadministration of interleukins 7 and 15 with bacille Calmette-Guérin mounts enduring T cell memory response against Mycobacterium tuberculosis.

BACKGROUND: The bacille Calmette-Guérin (BCG) vaccine renders protection against tuberculosis in childhood but not in adulthood. This may be due to its failure to induce long-lasting memory T cells. T cell memory is dependent on crucial cytokine signals during the priming phases. Therefore, coadministering the BCG vaccine with cytokines may improve its efficacy. METHODS: A combination of the cytokines interleukin 7 (IL-7) and interleukin 15 (IL-15) or a combination of the cytokines interleukin 1 (IL-1), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-alpha), which are known to influence memory T cell generation, were administered along with BCG to mice. The animals were rested for a period of 240 d before they were challenged with Mycobacterium tuberculosis. Five weeks later, they were killed to study the T cell memory response. RESULTS: Administration of IL-7 and IL-15, but not IL-1, IL-6, and TNF-alpha, with BCG resulted in an improved CD4 and CD8 T cell memory response. Mice injected with BCG supplemented with IL-7 and IL-15 showed enhanced T cell proliferation, T helper 1-type cytokine production, and an increased pool of multifunctional M. tuberculosis-specific memory T cells. Furthermore, there was a statistically significant reduction in the mycobacterial burden in the lungs. CONCLUSION: Our results indicate that supplementation of the BCG vaccine with IL-7 and IL-15 would substantially improve its efficacy by enhancing the T cell memory response.

Immunotherapy With 5, 15-DPP Mediates Macrophage M1 Polarization and Modulates Subsequent Mycobacterium tuberculosis Infectivity in rBCG30 Immunized Mice.

Tuberculosis (TB) is a significant and continuing problem worldwide, with a death toll of around 1.5 million human lives annually. BCG, the only vaccine against TB, offers a varied degree of protection among human subjects in different regions and races of the world. The majority of the population living near the tropics carries a varying degree of tolerance against BCG due to the widespread prevalence of non-tuberculous mycobacteria (NTM). Interestingly, ≈90% of the Mycobacterium tuberculosis (Mtb) infected population restrain the bacilli on its own, which strengthens the notion of empowering the host immune system to advance the protective efficacy of existing mycobacterial vaccines. In general, Mtb modulates IL-10/STAT3 signaling to skew host mononuclear phagocytes toward an alternatively activated, anti-inflammatory state that helps it thrive against hostile immune advances. We hypothesized that modulating the IL-10/STAT3 driven anti-inflammatory effects in mononuclear cells may improve the prophylactic ability of TB vaccines. This study investigated the immunotherapeutic ability of a porphyrin based small molecule inhibitor of IL-10/STAT3 axis, 5, 15-diphenyl porphyrin (DPP), in improving anti-TB immunity offered by second generation recombinant BCG30 (rBCG30-ARMF-II®) vaccine in mice. The DPP therapy potentiated vaccine induced anti-TB immunity by down-modulating anti-inflammatory responses, while simultaneously up-regulating pro-inflammatory immune effector responses in the immunized host. The employed DPP based immunotherapy led to the predominant activation/proliferation of pro-inflammatory monocytes/macrophages/DCs, the concerted expansion of CD4+/CD8+ effector and central memory T cells, alongside balanced Th17 and Treg cell amplification, and conferred augmented resistance to aerosol Mtb challenge in rBCG30 immunized BALB/c mice.

Inhalable particles containing isoniazid and rifabutin as adjunct therapy for safe, efficacious and relapse-free cure of experimental animal tuberculosis in one month.

We investigated the preclinical efficacy and safety/tolerability of biodegradable polymeric particles containing isoniazid (INH) and rifabutin (RFB) dry powder for inhalation (DPI) as an adjunct to oral first-line therapy. Mice and guinea pigs infected with Mycobacterium tuberculosis H37Rv (Mtb) were treated with ∼80 and ∼300 µg of the DPI, respectively, for 3-4 weeks starting 3, 10, and 30 days post-infection. Adjunct combination therapy eliminated culturable Mtb from the lungs and spleens of all but one of 52 animals that received the DPI. Relapse-free cure was not achieved in one mouse that received DPI + oral, human-equivalent doses (HED) of four drugs used in the Directly Observed Treatment, Short Course (DOTS), starting 30 days post-infection. Oral doses (20 mg/Kg/day, each) of INH + RFB reduced Mtb burden from ∼106 to ∼103 colony-forming units. Combining half the oral dose with DPI prevented relapse of infection four weeks after stopping the treatment. The DPI was safe in rodents, guinea pigs, and monkeys at 1, 10, and 100 µg/day doses over 90 days. In conclusion, we show the efficacy and safety/tolerability of the DPI as an adjunct to oral chemotherapy in three different animal models of TB.

Dynamic mucus penetrating microspheres for efficient pulmonary delivery and enhanced efficacy of host defence peptide (HDP) in experimental tuberculosis.

Pulmonary drug delivery system is increasingly gaining popularity for several lung diseases including tuberculosis(TB) due to its ability to attain high drug concentrations at the site of infection and to minimize systemic toxicity. In TB therapy, the efficacy of the antibiotics decreases and bacteria becomes resistant in course of time due to the formation of several barriers like lung-mucus and biofilms around the microorganism. The conventional inhalable microparticles(MP) are majorly trapped in dense mucin mess network and quickly cleared by mucocilliary clearance. In this study, we determined whether the anti-TB activity of drug-loaded inhalable polymeric microparticles could be synergized with the mucus-penetrating and biofilm disrupting properties. Mucus-penetrating-microparticles(NAC/PLGA-MPP) were developed combining the benefits of anti-TB drug with host defence peptides(HDP). IDR-1018 peptide was encapsulated with/without an anti-TB drug in N-acetyl cysteine(NAC) decorated porous PLGA microspheres. Aerodynamic parameters(MMAD-3.79 ± 1.04 µm, FPF-52.9 ± 5.11%) were optimized for the finest deposition and targeting inside the lungs. The multiple-tracking-technique(MPT) results indicate that the coating of NAC on porous PLGA-MS dramatically increased (4.1fold) the particle transit through the mucus barrier. Designed inhalable NAC/PLGA-MPP do not adhere to lung mucus, disrupt the bacterial biofilm and provide uniform drug delivery to lungs after pulmonary delivery. The formulation was evaluated for activity against M.tb in macrophage cultures and in mice model infected with a low-dose bacterial (~100 CFU) aerosol. The inhalation of NAC/PLGA-MPP encapsulated with IDR-1018 significantly reduced (p < .05) bacterial load (up to ~3.02LogCFU/ml) and inflammation in lungs in a mouse model of TB compared to untreated and blank treated animals in 6 weeks of daily dose. The histopathological results validate the compelling chemotherapeutic outcome of inhaled formulations. This data supports the harnessing potential of mucus penetrating inhalable drug delivery systems as a vehicle for targeted lung delivery. This "value-added" inhalable formulation could be beneficial for resistant TB therapeutics when used as an "adjunct" to existing DOTS (Directly observed treatment, short-course) therapy.