Efficacy of macozinone in mice with genetically diverse susceptibility to Mycobacterium tuberculosis infection.

Host heterogeneity in pulmonary tuberculosis leads to varied responses to infection and drug treatment. The present portfolio of anti-TB drugs needs to be boosted with new drugs and drug regimens. Macozinone, a clinical-stage molecule targeting the essential enzyme, DprE1, represents an attractive option. Mice (I/St, B6, (AKRxI/St)F1, B6.I-100 and B6.I-139) genetically diverse susceptibility to Mycobacterium tuberculosis (Mtb) H37Rv infection were subjected to aerosol- or intravenous infection to determine the efficacy of macozinone (MCZ). They were treated with macozinone or reference drugs (isoniazid, rifampicin). Lung and spleen bacterial burdens were measured at four and eight weeks post-infection. Lung histology was evaluated at four weeks of treatment. Treatment with macozinone resulted in a statistically significant reduction in the bacterial load in the lungs and spleen as early as four weeks after treatment initiation in mice susceptible or resistant to Mtb infection. In the TB hypoxic granuloma model, macozinone was more potent than rifampicin in reducing the CFU counts. However, histopathological analysis revealed significant lung changes in I/St mice after eight weeks of treatment initiation. Macozinone demonstrated efficacy to varying degrees across all mouse models of Mtb infection used. These results should facilitate its further development and potential introduction into clinical practice.

Metabolic Fate of Human Immunoactive Sterols in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) infection is among top ten causes of death worldwide, and the number of drug-resistant strains is increasing. The direct interception of human immune signaling molecules by Mtb remains elusive, limiting drug discovery. Oxysterols and secosteroids regulate both innate and adaptive immune responses. Here we report a functional, structural, and bioinformatics study of Mtb enzymes initiating cholesterol catabolism and demonstrated their interrelation with human immunity. We show that these enzymes metabolize human immune oxysterol messengers. Rv2266 - the most potent among them - can also metabolize vitamin D3 (VD3) derivatives. High-resolution structures show common patterns of sterols binding and reveal a site for oxidative attack during catalysis. Finally, we designed a compound that binds and inhibits three studied proteins. The compound shows activity against Mtb H37Rv residing in macrophages. Our findings contribute to molecular understanding of suppression of immunity and suggest that Mtb has its own transformation system resembling the human phase I drug-metabolizing system.

Structural Modifications of 3-Triazeneindoles and Their Increased Activity Against Mycobacterium tuberculosis.

We synthesized 100 novel indole-based compounds with polyaza-functionalities, including 3-triazeneindoles, and tested their activity in vitro against laboratory M. tuberculosis H37Rv and clinical izoniazid-resistant CN-40 isolates, using gross and fine titration approaches. Here we present a few 3-triazeneindoles with the highest anti-mycobacterial activity. Introduction of short lipid tails into the 3-triazeneindole core additionally increased their activity against mycobacteria engulfed by murine macrophages. We also demonstrate that the compound TU112, one of the most active in our previous study, being not bioavailable after administration in mice per os, manifests prominent anti-mycobacterial activity after intravenous or aerosol delivery, as assessed by the mouse serum and lung supernatant titration assays.

A new model for chronic and reactivation tuberculosis: Infection with genetically attenuated Mycobacterium tuberculosis in mice with polar susceptibility.

TB infection in mice develops relatively rapidly which interferes with experimental dissection of immune responses and lung pathology features that differ between genetically susceptible and resistant hosts. Earlier we have shown that the M. tuberculosis strain lacking four of five Rpf genes (ΔACDE) is seriously attenuated for growth in vivo. Using this strain, we assessed key parameters of lung pathology, immune and inflammatory responses in chronic and reactivation TB infections in highly susceptible I/St and more resistant B6 mice. ΔACDE mycobacteria progressively multiplied only in I/St lungs, whilst in B6 lung CFU counts decreased with time. Condensed TB foci apeared in B6 lungs at week 4 of infection, whilst in I/St their formation was delayed. At the late phase of infection, in I/St lungs TB foci fused resulting in extensive pneumonia, whereas in B6 lungs pathology was limited to condensed foci. Macrophage and neutrophil populations characteristically differed between I/St and B6 mice at early and late stages of infection: more neutrophils accumulated in I/St and more macrophages in B6 lungs. The expression level of chemokine genes involved in neutrophil influx was higher in I/St compared to B6 lungs. B6 lung cells produced more IFN-γ, IL-6 and IL-11 at the early and late phases of infection. Overall, using a new mouse model of slow TB progression, we demonstrate two important features of ineffective infection control underlined by shifts in lung inflammation: delay in early granuloma formation and fusion of granulomas resulting in consolidated pneumonia late in the infectious course.

New 1-hydroxy-2-thiopyridine derivatives active against both replicating and dormant Mycobacterium tuberculosis.

Tuberculosis (TB) treatment is confounded by the range of metabolic states displayed by Mycobacterium tuberculosis, by the long duration required and by the increasing prevalence of drug-resistant strains. Latent TB infection is especially difficult to treat due to the phenotypic antibiotic resistance of non-replicating M. tuberculosis. Therefore, the development of new drugs effective against both active and latent TB infection is needed. New 1-hydroxy-2-thiopyridine derivatives were synthesized and found to be highly effective in vitro against both actively growing and dormant non-culturable M. tuberculosis. Such compounds are leads for the development of new drugs for all forms of TB including latent infection.

In Vitro Activity of 3-Triazeneindoles against Mycobacterium tuberculosis and Mycobacterium avium.

Among 230 target-synthesized indole-based compounds, seven 3-triazenoindoles showed MICs of 0.2 to 0.5 µg/ml against Mycobacterium tuberculosis strain H37Rv and isoniazid-resistant human isolate CN-40. The TU112 compound was active also against a dormant form of M. tuberculosis Some of these triazenoindoles were active against Mycobacterium avium, with MICs of 0.05 to 0.5 µg/ml. The selectivity indices (SI) for M. tuberculosis and M. avium were significantly higher than 10, making these compounds acceptable for the next testing step.

Treatment of Clostridium difficile infection using SQ641, a capuramycin analogue, increases post-treatment survival and improves clinical measures of disease in a murine model.

OBJECTIVES: Clostridium difficile infection (CDI) is a primary cause of antibiotic-associated diarrhoeal illness. Current therapies are insufficient as relapse rates following antibiotic treatment range from 25% for initial treatment to 60% for treatment of recurrence. In this study, we looked at the efficacy of SQ641 in a murine model of CDI. SQ641 is an analogue of capuramycin, a naturally occurring nucleoside-based compound produced by Streptomyces griseus. METHODS: In a series of experiments, C57BL/6 mice were treated with a cocktail of antibiotics and inoculated with C. difficile strain VPI10463. Animals were treated orally with SQ641 for 5 days at a dose range of 0.1-300 mg/kg/day, 20 mg/kg/day vancomycin or drug vehicle. Animals were monitored for disease severity, clostridial shedding and faecal toxin levels for 14 days post-infection. RESULTS: Five day treatment of CDI with SQ641 resulted in higher 14 day survival rates in mice compared with either vancomycin or vehicle alone. CDI survival rates were 100% (13 of 13) and 94% (32 of 34), respectively, in the 1 and 10 mg/kg/day SQ641 treatment groups, 37% (7 of 19) with vancomycin treatment at 20 mg/kg/day and 32% (14 of 44) in the vehicle-only control group. Secondary measures of efficacy, such as prevention of weight loss, decreased disease severity, decreased C. difficile shedding and decreased toxin in faeces, were observed with SQ641 and vancomycin treatment. CONCLUSIONS: SQ641 is effective for CDI treatment with prevention of relapse in the murine model of CDI.

Synthesis and antituberculosis activity of indole-pyridine derived hydrazides, hydrazide-hydrazones, and thiosemicarbazones.

We describe the design, synthesis, and in vitro antimycobacterial activity of a series of novel simple hybrid hydrazides and hydrazide-hydrazones combining indole and pyridine nuclei. The compounds are derivatives of 1-acetylindoxyl or substituted indole-3-carboxaldehydes tethered via a hydrazine group by simple C-N or double C=N bonds with 3- and 4-pyridines, 1-oxide 3- and 4-pyridine carbohydrazides. The most active of 15 compounds showed MICs values against an INH-sensitive strain of Mycobacterium tuberculosis H37Rv equal to that of INH (0.05-2 µg/mL). Five compounds demonstrated appreciable activity against the INH-resistant M. tuberculosis CN-40 clinical isolate (MICs: 2-5 µg/mL), providing justification for further in vivo studies.

Latent tuberculosis infection: what we know about its genetic control?

About 90% of all cases of tuberculosis (TB) infection are comprised of latent mycobacterial persistence in the absence of clinical manifestations. In a proportion of latently infected individuals infection eventually reactivates and becomes contagious, seriously influencing epidemiological situation. Mechanisms of Mycobacterium tuberculosis transition to dormancy and TB reactivation are poorly understood, and biological markers of latency remain largely unknown. Data are accumulating that the dynamical equilibrium between the parasite and the host (expressed as a long term asymptomatic infection) and its abrogation (expressed as a reactivation disease) are genetically controlled by both parties. In this short review, the authors summarize the results of experimental studies on genetic regulation of the latent TB infection.

Therapeutic efficacy of SQ641-NE against Mycobacterium tuberculosis.

A phospholipid-based nanoemulsion formulation of SQ641 (SQ641-NE) was active against intracellular Mycobacterium tuberculosis in J774A.1 mouse macrophages, although SQ641 by itself was not. Intravenous (i.v.) SQ641-NE was cleared from circulation and reached peak concentrations in lung and spleen in 1 h. In a murine tuberculosis (TB) model, 8 i.v. doses of SQ641-NE at 100 mg/kg of body weight over 4 weeks caused a 1.73 log10 CFU reduction of M. tuberculosis in spleen and were generally bacteriostatic in lungs.

Drug testing in mouse models of tuberculosis and nontuberculous mycobacterial infections.

Mice as a species are susceptible to tuberculosis infection while mouse inbred strains present wide spectrum of susceptibility/resistance to this infection. However, non-tuberculosis Mycobacterial infections usually cannot be modeled in mice of common inbred strains. Introduction of specific properties, such as gene mutations, recombinants, targeted gene knockouts significantly extended the use of mice to mimic human Mycobacterial infections, including non-tuberculosis ones. This review describes the available mouse models of tuberculosis and non-tuberculosis infections and drug therapy in these models. Mouse models of non-tuberculosis infections are significantly less developed than tuberculosis models, hampering the development of therapies.

Chronic helminth infection does not exacerbate Mycobacterium tuberculosis infection.

BACKGROUND: Chronic helminth infections induce a Th2 immune shift and establish an immunoregulatory milieu. As both of these responses can suppress Th1 immunity, which is necessary for control of Mycobacterium tuberculosis (MTB) infection, we hypothesized that chronic helminth infections may exacerbate the course of MTB. METHODOLOGY/PRINCIPAL FINDINGS: Co-infection studies were conducted in cotton rats as they are the natural host for the filarial nematode Litomosoides sigmodontis and are an excellent model for human MTB. Immunogical responses, histological studies, and quantitative mycobacterial cultures were assessed two months after MTB challenge in cotton rats with and without chronic L. sigmodontis infection. Spleen cell proliferation and interferon gamma production in response to purified protein derivative were similar between co-infected and MTB-only infected animals. In contrast to our hypothesis, MTB loads and occurrence and size of lung granulomas were not increased in co-infected animals. CONCLUSIONS/SIGNIFICANCE: These findings suggest that chronic filaria infections do not exacerbate MTB infection in the cotton rat model. While these results suggest that filaria eradication programs may not facilitate MTB control, they indicate that it may be possible to develop worm-derived therapies for autoimmune diseases that do not substantially increase the risk for infections.

Identification of SQ609 as a lead compound from a library of dipiperidines.

We recently reported that compounds created around a dipiperidine scaffold demonstrated activity against Mycobacterium tuberculosis (Mtb) (Bogatcheva, E.; Hanrahan, C.; Chen, P.; Gearhart, J.; Sacksteder, K.; Einck, L.; Nacy, C.; Protopopova, M. Bioorg. Med. Chem. Lett.2010, 20, 201). To optimize the dipiperidine compound series and to select a lead compound to advance into preclinical studies, we evaluated the structure-activity relationship (SAR) of our proprietary libraries. The (piperidin-4-ylmethyl)piperidine scaffold was an essential structural element required for antibacterial activity. Based on SAR, we synthesized a focused library of 313 new dipiperidines to delineate additional structural features responsible for antitubercular activity. Thirty new active compounds with MIC 10-20 µg/ml on Mtb were identified, but none was better than the original hits of this series, SQ609, SQ614, and SQ615. In Mtb-infected macrophages in vitro, SQ609 and SQ614 inhibited more than 90% of intracellular bacterial growth at 4 µg/ml; SQ615 was toxic to these cells. In mice infected with Mtb, weight loss was completely prevented by SQ609, but not SQ614, and SQ609 had a prolonged therapeutic effect, extended by 10-15 days, after cessation of therapy. Based on in vitro and in vivo antitubercular activity, SQ609 was identified as the best-in-class dipiperidine compound in the series.

Anti-tuberculosis drug therapy in mice of different inbred strains.

Standard anti-tuberculosis (TB) drug therapy had distinct effects on the bacilli burden in mice of DBA/2, C3H, SWR/J, and C57BL/6 inbred strains. To standardize the TB infection process, susceptible DBA/2 mice were infected with 1/10 of the dose used for relatively resistant C57BL/6 mice, such that the lung CFUs were roughly identical 3 weeks after infection when therapy was initiated. We found that TB treatment was more effective in the susceptible DBA/2 mice than in the relatively resistant C57BL/6 mice.

Activity of SQ641, a capuramycin analog, in a murine model of tuberculosis.

New delivery vehicles and routes of delivery were developed for the capuramycin analogue SQ641. While this compound has remarkable in vitro potency against Mycobacterium tuberculosis, it has low solubility in water and poor intracellular activity. We demonstrate here that SQ641 dissolved in the water-soluble vitamin E analogue alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) or incorporated into TPGS-micelles has significant activity in a mouse model of tuberculosis.

Characterization of late tuberculosis infection in Sigmodon hispidus.

We previously described primary tuberculosis in Sigmodon hispidus cotton rats up to 6 months following a pulmonary challenge. At that time, we observed fewer animals demonstrating disease as time from exposure progressed. We hypothesized that some cotton rats may control a primary infection to latency in a similar fashion to humans. The current experiment was designed to examine the natural progression of disease in S. hispidus at a later timepoint following a respiratory challenge with Mycobacterium tuberculosis (Mtb). An additional objective was to test whether cotton rats may become latently infected, and to determine whether latent disease might be activated by cyclophosphamide induced immune suppression. Thirty-four percent of the inoculated cotton rats died prior to 9 months following the challenge. However, 50% of immunocompetent animals surviving past 9 months demonstrated positive lung tissue cultures for Mtb without histologic evidence of disease. None of the immunosuppressed animals demonstrated this pattern. These findings are consistent with the development of latent tuberculosis infection in some cotton rats. Furthermore, it appears reactivation of disease occurs with cyclophosphamide induced immunosuppression. Cotton rats may serve as a model for latent as well as active tuberculosis infection.

Preclinical study of new TB drugs and drug combinations in mouse models.

Tuberculosis (TB) remains one of the leading infectious killers in the world. New anti-TB drugs and more effective drug combinations are urgently needed, particularly given the increasing incidence of drug-resistant TB and HIV-TB co-infection. This review describes the available mouse models of TB and describes their utility in the evaluation of new TB drug candidates and in the evaluation of the efficacy of new TB drug combinations. Some of the most recent patents on promising TB drug-candidates are also mentioned here.

In search of new cures for tuberculosis.

The last 10 years have seen resurgent industry activity in discovery and development of new drugs for the treatment of tuberculosis (TB), a growing widespread and devastating (more than 2 million deaths annually) bacterial infection that is of increasing concern in developing and developed nations alike. This review describes drugs currently being evaluated in the clinic for treatment of uncomplicated and drug resistant pulmonary TB, and updates the literature on 5 new drugs that entered clinical trials in the last 4 years.

Drug therapy of experimental tuberculosis (TB): improved outcome by combining SQ109, a new diamine antibiotic, with existing TB drugs.

Substitution of the new diamine antibiotic SQ109 for ethambutol in a mouse model of chronic tuberculosis (TB) improved efficacy of combination drug therapy with first-line TB drugs rifampin and isoniazid, with or without pyrazinamide: at 8 weeks, lung bacteria were 1.5 log10 lower in SQ109-containing regimens.

The American cotton rat: a novel model for pulmonary tuberculosis.

Several animal models are used to study Mycobacterium tuberculosis (MTB) infections, but none is a fully ideal model of human disease. The American cotton rat is an excellent model for the study of several human viral and bacterial respiratory infectious diseases, but until now has not been reported to be a model with MTB infection. Preliminary experiments were designed in which two species of cotton rats (Sigmodon hispidus and Sigmodon fulviventer) received respiratory challenges with M. tuberculosis via either intranasal or aerosol inoculation. Granulomatous disease, often with central necrosis, developed in the lungs, spleen, and lymph nodes of infected animals. The number of MTB bacilli in the lungs increased logarithmically until reaching a plateau in the second month after aerosol inoculation. There were differences in response to infection between the two species, with S. fulviventer demonstrating greater mortality than S. hispidus. Cytokine gene expression analysis by reverse transcriptase polymerase chain reaction (RT-PCR) was performed on both normal appearing and granulomatous lung tissue from infected animals. Many cytokine genes were more highly expressed in the focal areas of inflammation. Cotton rats provide another valuable tool in future research with tuberculosis.