RESUMEN
Multiple drug discovery initiatives for tuberculosis are currently ongoing to identify and develop new potent drugs with novel targets in order to shorten treatment duration. One of the drug classes with a new mode of action is DprE1 inhibitors targeting an essential process in cell wall synthesis of Mycobacterium tuberculosis. In this investigation, three DprE1 inhibitors currently in clinical trials, TBA-7371, PBTZ169, and OPC-167832, were evaluated side-by-side as single agents in the C3HeB/FeJ mouse model presenting with caseous necrotic pulmonary lesions upon tuberculosis infection. The goal was to confirm the efficacy of the DprE1 inhibitors in a mouse tuberculosis model with advanced pulmonary pathology and perform comprehensive analysis of plasma, lung, and lesion-centric drug levels to establish pharmacokinetic-pharmacodynamic (PK-PD) parameters predicting efficacy at the site of infection. Results showed significant efficacy for all three DprE1 inhibitors in the C3HeB/FeJ mouse model after 2 months of treatment. Superior efficacy was observed for OPC-167832 even at low-dose levels, which can be attributed to its low MIC, favorable distribution, and sustained retention above the MIC throughout the dosing interval in caseous necrotic lesions, where the majority of bacteria reside in C3HeB/FeJ mice. These results support further progression of the three drug candidates through clinical development for tuberculosis treatment.
Asunto(s)
Mycobacterium tuberculosis , Tiazinas , Tuberculosis , Animales , Ratones , Ratones Endogámicos C3H , Piperazinas , Tuberculosis/tratamiento farmacológicoRESUMEN
New antitubercular agents are needed to combat the spread of multidrug- and extensively drug-resistant strains of Mycobacterium tuberculosis. The frontline antitubercular drug isoniazid (INH) targets the mycobacterial enoyl-ACP reductase, InhA. Resistance to INH is predominantly through mutations affecting the prodrug-activating enzyme KatG. Here, we report the identification of the diazaborines as a new class of direct InhA inhibitors. The lead compound, AN12855, exhibited in vitro bactericidal activity against replicating bacteria and was active against several drug-resistant clinical isolates. Biophysical and structural investigations revealed that AN12855 binds to and inhibits the substrate-binding site of InhA in a cofactor-independent manner. AN12855 showed good drug exposure after i.v. and oral delivery, with 53% oral bioavailability. Delivered orally, AN12855 exhibited dose-dependent efficacy in both an acute and chronic murine model of tuberculosis infection that was comparable with INH. Combined, AN12855 is a promising candidate for the development of new antitubercular agents.
RESUMEN
The recent development and spread of extensively drug-resistant and totally drug-resistant resistant (TDR) strains of Mycobacterium tuberculosis highlight the need for new antitubercular drugs. Protein synthesis inhibitors have played an important role in the treatment of tuberculosis (TB) starting with the inclusion of streptomycin in the first combination therapies. Although parenteral aminoglycosides are a key component of therapy for multidrug-resistant TB, the oxazolidinone linezolid is the only orally available protein synthesis inhibitor that is effective against TB. Here, we show that small-molecule inhibitors of aminoacyl-tRNA synthetases (AARSs), which are known to be excellent antibacterial protein synthesis targets, are orally bioavailable and effective against M. tuberculosis in TB mouse infection models. We applied the oxaborole tRNA-trapping (OBORT) mechanism, which was first developed to target fungal cytoplasmic leucyl-tRNA synthetase (LeuRS), to M. tuberculosis LeuRS. X-ray crystallography was used to guide the design of LeuRS inhibitors that have good biochemical potency and excellent whole-cell activity against M. tuberculosis Importantly, their good oral bioavailability translates into in vivo efficacy in both the acute and chronic mouse models of TB with potency comparable to that of the frontline drug isoniazid.
Asunto(s)
Antituberculosos/farmacología , Leucina-ARNt Ligasa/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Administración Oral , Animales , Antituberculosos/administración & dosificación , Antituberculosos/química , Antituberculosos/farmacocinética , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos/métodos , Femenino , Humanos , Leucina-ARNt Ligasa/química , Leucina-ARNt Ligasa/genética , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos , Pruebas de Sensibilidad Microbiana , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Inhibidores de la Síntesis de la Proteína/administración & dosificación , Inhibidores de la Síntesis de la Proteína/química , Inhibidores de la Síntesis de la Proteína/farmacocinética , Relación Estructura-Actividad , Tuberculosis/tratamiento farmacológico , Células VeroRESUMEN
New drugs and drugs with a novel mechanism of action are desperately needed to shorten the duration of tuberculosis treatment, to prevent the emergence of drug resistance, and to treat multiple-drug-resistant strains of Mycobacterium tuberculosis. Recently, there has been renewed interest in clofazimine (CFZ). In this study, we utilized the C3HeB/FeJ mouse model, possessing highly organized, hypoxic pulmonary granulomas with caseous necrosis, to evaluate CFZ monotherapy in comparison to results with BALB/c mice, which form only multifocal, coalescing cellular aggregates devoid of caseous necrosis. While CFZ treatment was highly effective in BALB/c mice, its activity was attenuated in the lungs of C3HeB/FeJ mice. This lack of efficacy was directly related to the pathological progression of disease in these mice, since administration of CFZ prior to the formation of hypoxic, necrotic granulomas reconstituted bactericidal activity in this mouse strain. These results support the continued use of mouse models of tuberculosis infection which exhibit a granulomatous response in the lungs that more closely resembles the pathology found in human disease.
Asunto(s)
Antituberculosos/uso terapéutico , Clofazimina/uso terapéutico , Granuloma/tratamiento farmacológico , Tuberculosis/tratamiento farmacológico , Animales , Carga Bacteriana , Modelos Animales de Enfermedad , Femenino , Granuloma/patología , Interferón gamma/genética , Interferón gamma/fisiología , Pulmón/microbiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Noqueados , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Necrosis , Tuberculosis/complicacionesRESUMEN
A series of tetracyclic nitrofuran isoxazoline anti-tuberculosis agents was designed and synthesized to improve the pharmacokinetic properties of an initial lead compound, which had potent anti-tuberculosis activity but suffered from poor solubility, high protein binding and rapid metabolism. In this study, structural modifications were carried on the outer phenyl and piperidine rings to introduce solubilizing and metabolically blocking functional groups. The compounds generated were evaluated for their in vitro antitubercular activity, bacterial spectrum of activity, solubility, permeability, microsomal stability and protein binding. Pharmacokinetic profiles for the most promising candidates were then determined. Compounds with phenyl morpholine and pyridyl morpholine outer rings were found to be the most potent anti-tuberculosis agents in the series. These compounds retained a narrow antibacterial spectrum of activity, with weak anti-Gram positive and no Gram negative activity, as well as good activity against non-replicating Mycobacterium tuberculosis in a low oxygen model. Overall, the addition of solubilizing and metabolically blocked outer rings did improve solubility and decrease protein binding as designed. However, the metabolic stability for compounds in this series was generally lower than desired. The best three compounds selected for in vivo pharmacokinetic testing all showed high oral bioavailability, with one notable compound showing a significantly longer half-life and good tolerability supporting its further advancement.
Asunto(s)
Antituberculosos/química , Nitrofuranos/química , Animales , Antituberculosos/síntesis química , Antituberculosos/farmacocinética , Células CACO-2 , Supervivencia Celular/efectos de los fármacos , Semivida , Humanos , Isoxazoles/química , Ratones , Pruebas de Sensibilidad Microbiana , Microsomas/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Nitrofuranos/síntesis química , Nitrofuranos/farmacocinética , Ratas , Solubilidad , Relación Estructura-ActividadRESUMEN
New chemotherapeutics active against multidrug-resistant Mycobacterium tuberculosis are urgently needed. We report on the identification of an adamantyl urea compound that shows potent bactericidal activity against M. tuberculosis and a unique mode of action, namely the abolition of the translocation of mycolic acids from the cytoplasm, where they are synthesized to the periplasmic side of the plasma membrane and are in turn transferred onto cell wall arabinogalactan or used in the formation of virulence-associated, outer membrane, trehalose-containing glycolipids. Whole-genome sequencing of spontaneous-resistant mutants of M. tuberculosis selected in vitro followed by genetic validation experiments revealed that our prototype inhibitor targets the inner membrane transporter MmpL3. Conditional gene expression of mmpL3 in mycobacteria and analysis of inhibitor-treated cells validate MmpL3 as essential for mycobacterial growth and support the involvement of this transporter in the translocation of trehalose monomycolate across the plasma membrane.
Asunto(s)
Adamantano/análogos & derivados , Antibacterianos/química , Antibacterianos/farmacología , Membrana Celular/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Ácidos Micólicos/metabolismo , Compuestos de Fenilurea/farmacología , Adamantano/química , Adamantano/farmacología , Antibacterianos/farmacocinética , Proteínas Bacterianas/metabolismo , Transporte Biológico/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Factores Cordón , Evaluación Preclínica de Medicamentos/métodos , Farmacorresistencia Bacteriana , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Compuestos de Fenilurea/química , Bibliotecas de Moléculas Pequeñas , Trehalosa/metabolismoRESUMEN
In preclinical testing of antituberculosis drugs, laboratory-adapted strains of Mycobacterium tuberculosis are usually used both for in vitro and in vivo studies. However, it is unknown whether the heterogeneity of M. tuberculosis stocks used by various laboratories can result in different outcomes in tests of antituberculosis drug regimens in animal infection models. In head-to-head studies, we investigated whether bactericidal efficacy results in BALB/c mice infected by inhalation with the laboratory-adapted strains H37Rv and Erdman differ from each other and from those obtained with clinical tuberculosis strains. Treatment of mice consisted of dual and triple drug combinations of isoniazid (H), rifampin (R), and pyrazinamide (Z). The results showed that not all strains gave the same in vivo efficacy results for the drug combinations tested. Moreover, the ranking of HRZ and RZ efficacy results was not the same for the two H37Rv strains evaluated. The magnitude of this strain difference also varied between experiments, emphasizing the risk of drawing firm conclusions for human trials based on single animal studies. The results also confirmed that the antagonism seen within the standard HRZ regimen by some investigators appears to be an M. tuberculosis strain-specific phenomenon. In conclusion, the specific identity of M. tuberculosis strain used was found to be an important variable that can change the apparent outcome of in vivo efficacy studies in mice. We highly recommend confirmation of efficacy results in late preclinical testing against a different M. tuberculosis strain than the one used in the initial mouse efficacy study, thereby increasing confidence to advance potent drug regimens to clinical trials.
Asunto(s)
Antituberculosos/uso terapéutico , Isoniazida/uso terapéutico , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/efectos de los fármacos , Pirazinamida/uso terapéutico , Rifampin/uso terapéutico , Tuberculosis Pulmonar/tratamiento farmacológico , Animales , Antituberculosos/farmacología , Modelos Animales de Enfermedad , Quimioterapia Combinada , Femenino , Humanos , Isoniazida/farmacología , Pulmón/microbiología , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana/estadística & datos numéricos , Mycobacterium tuberculosis/genética , Pirazinamida/administración & dosificación , Pirazinamida/farmacología , Rifampin/administración & dosificación , Rifampin/farmacología , Resultado del Tratamiento , Tuberculosis Pulmonar/microbiologíaRESUMEN
Methodologies for preclinical animal model testing of drugs against Mycobacterium tuberculosis vary from laboratory to laboratory; however, it is unknown if these variations result in different outcomes. Thus, a series of head-to-head comparisons of drug regimens in three commonly used mouse models (intravenous, a low-dose aerosol, and a high-dose aerosol infection model) and in two strains of mice are reported here. Treatment with standard tuberculosis (TB) drugs resulted in similar efficacies in two mouse species after a low-dose aerosol infection. When comparing the three different infection models, the efficacies in mice of rifampin and pyrazinamide were similar when administered with either isoniazid or moxifloxacin. Relapse studies revealed that the standard drug regimen showed a significantly higher relapse rate than the moxifloxacin-containing regimen. In fact, 4 months of the moxifloxacin-containing combination regimen showed similar relapse rates as 6 months of the standard regimen. The intravenous model showed slower bactericidal killing kinetics with the combination regimens tested and a higher relapse of infection than either aerosol infection models. All three models showed similar outcomes for in vivo efficacy and relapse of infection for the drug combinations tested, regardless of the mouse infection model used. Efficacy data for the drug combinations used also showed similar results, regardless of the formulation used for rifampin or timing of the drugs administered in combination. In all three infection models, the dual combination of rifampin and pyrazinamide was less sterilizing than the standard three-drug regimen, and therefore the results do not support the previously reported antagonism between standard TB agents.
Asunto(s)
Antituberculosos/uso terapéutico , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/patogenicidad , Tuberculosis/tratamiento farmacológico , Animales , Quimioterapia Combinada , Femenino , Isoniazida/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Pirazinamida/uso terapéutico , Rifampin/uso terapéutico , Tuberculosis/microbiologíaRESUMEN
A novel subclass of quinolones, 2-pyridones, showed potent activity against Mycobacterium tuberculosis, with KRQ-10018 being an early lead. KRQ-10018 showed better activity in vitro against M. tuberculosis versus moxifloxacin. In vivo efficacy of KRQ-10018 at 300 mg/kg of body weight was similar to that of isoniazid at 25 mg/kg, but showed less activity than moxifloxacin at 300 mg/kg.
Asunto(s)
Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Mycobacterium tuberculosis/efectos de los fármacos , Piridonas/farmacología , Piridonas/uso terapéutico , Tuberculosis Pulmonar/tratamiento farmacológico , Animales , Antituberculosos/administración & dosificación , Disponibilidad Biológica , Interferón gamma/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pruebas de Sensibilidad Microbiana , Piridonas/administración & dosificación , Quinolonas/administración & dosificación , Quinolonas/farmacología , Quinolonas/uso terapéutico , Resultado del Tratamiento , Tuberculosis Pulmonar/microbiologíaRESUMEN
Structure-based design was used to develop a focused library of A-ring-modified diphenyl ether InhA inhibitors. From this library of analogs, two high-affinity alkyl-substituted diphenyl ethers, 6PP and 8PP, were selected for advanced study into their in vitro activity against Mycobacterium tuberculosis clinical isolates, their in vivo properties, and their signature response mode of action. 6PP and 8PP demonstrated enhanced activity against whole bacteria and showed activity in a rapid macrophage model of infection. In addition, transcriptional profiling revealed that the A-ring modifications of 6PP and 8PP increased the specificity of each analog for InhA. Both analogs had substantially longer half-lives in serum than did the parent compound, exhibited a fivefold reduction in cytotoxicity compared to the parent compound, and were well tolerated when administered orally at 300 mg/kg of body weight in animal models. Thus, the A-ring modifications increased the affinity and whole-cell specificity of the compounds for InhA and increased their bioavailability. The next step in optimization of the pharmacophore for preclinical evaluation is modification of the B ring to increase the bioavailability to that required for oral delivery.
Asunto(s)
Antituberculosos/síntesis química , Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Ácidos Grasos/biosíntesis , Mycobacterium tuberculosis/efectos de los fármacos , Oxidorreductasas/antagonistas & inhibidores , Éteres Fenílicos/farmacología , Animales , Proteínas Bacterianas/genética , Disponibilidad Biológica , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Dermatoglifia del ADN , Diseño de Fármacos , Farmacorresistencia Bacteriana , Femenino , Humanos , Técnicas In Vitro , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/genética , Oxidorreductasas/genética , Fagocitosis/efectos de los fármacos , Relación Estructura-Actividad , Transcripción Genética/efectos de los fármacos , Triclosán/farmacología , Células VeroRESUMEN
Cultures of Mycobacterium tuberculosis grown under oxygen depletion conditions enter into a state of nonreplicating persistence that may reflect a physiologically latent state. When these cultures were harvested and injected intranasally into mice, no bacteria could be recovered from the lungs for about 3 weeks, but after that evidence of regrowth was observed. Preimmunization of mice with a panel of selected vaccine candidates slowed or prevented this event. This simple model has potential for identifying vaccines targeting latent tuberculosis.
Asunto(s)
Adaptación Fisiológica , Modelos Animales de Enfermedad , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/patogenicidad , Oxígeno/farmacología , Tuberculosis Pulmonar/patología , Animales , Femenino , Pulmón/microbiología , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/fisiología , Organismos Libres de Patógenos Específicos , Tuberculosis Pulmonar/microbiologíaRESUMEN
The genetic region of difference 1 (RD1) in Mycobacterium tuberculosis has recently been hypothesized to encode for proteins that are cytotoxic to the host cell in nature. We demonstrate here that while M. tuberculosis grew progressively in the lungs of gene disrupted mice (GKO) unable to produce interferon-gamma (IFN-gamma), similar mice infected instead with M. bovis bacillus Calmette-Guérin (BCG) reproducibly exhibited an obvious slowing of the disease after about 20 days. Closer examination of BCG-infected GKO mice showed a florid granulomatous inflammation in the lungs, whereas similar mice infected with M. tuberculosis exhibited wholesale progressive necrosis. In the BCG-infected GKO mice large numbers of activated effector T cells, some strongly positive for the cytokine tumour necrosis factor, as well as activated natural killer cells accumulated in the lungs. To further test the hypothesis that the differences observed were directly associated with the loss of the RD1 region, it was then shown that a mutant of M. tuberculosis lacking RD1 grew progressively in both normal and GKO mice but failed to induce any degree of necrosis in either animal despite reaching similar levels in the lungs. However, when mice were infected with this mutant, in which the RD1 region had been restored by complementation, wholesale necrosis of the lungs again occurred. These data support the hypothesis that proteins encoded in the RD1 region are a major cause of necrosis and contribute significantly to the pathogenesis of the disease.
Asunto(s)
Interferón gamma/inmunología , Pulmón/patología , Mycobacterium bovis/patogenicidad , Mycobacterium tuberculosis/patogenicidad , Tuberculosis/inmunología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Movimiento Celular/inmunología , Progresión de la Enfermedad , Femenino , Genes Bacterianos , Interferón gamma/deficiencia , Interferón gamma/genética , Pulmón/microbiología , Activación de Linfocitos/inmunología , Activación de Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mycobacterium bovis/genética , Mycobacterium bovis/crecimiento & desarrollo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Necrosis , Tuberculosis/microbiología , VirulenciaRESUMEN
The assessment of physiochemical and pharmacological properties at early stages of drug discovery can accelerate the conversion of hits and leads into candidates for further development. A strategy for streamlined evaluation of compounds against Mycobacterium tuberculosis in the early preclinical stage is presented in this report. As a primary assay to rapidly select experimental compounds with sufficient in vitro activity, the growth inhibition microtiter plate assay was devised as an alternative to current methods. This microdilution plate assay is a liquid culture method based on spectrophotometric readings of the bacillary growth. The performance of this method was compared to the performance of two established susceptibility methods using clinical available tuberculosis (TB) drugs. Data generated from all three assays were similar for all of the tested compounds. A second simple bioassay was devised to assess the oral bioavailability of compounds prior to extensive in vivo efficacy testing. The bioassay estimates drug concentrations in collected serum samples by a microdilution MIC plate method using M. tuberculosis. In the same assay, the MIC of the compound is also determined in the presence of 10% mouse serum as an indication of protein binding. The method was validated using different clinically available TB drugs, and results are discussed in this report. With these methodological advances, screening of compounds against tuberculosis in the preclinical phase will be rapid, can be adapted to semi-high-throughput screening, and will add relevant physicochemical and basic pharmacological criteria to the decision process of drug discovery.
Asunto(s)
Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Animales , Antituberculosos/farmacocinética , Disponibilidad Biológica , Femenino , Ratones , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Unión ProteicaRESUMEN
Besides the long-term effectiveness of a given compound, safety is a very important feature to consider when developing new compounds for chemotherapy against tuberculosis. Reports of fatal and severe liver injury associated with rifampicin-pyrazinamide (RIF-PZA) treatment regimens for latent tuberculosis infections prompted this study to evaluate whether a mouse model has any potential as a tool to assess liver injury following extensive exposure to tuberculosis drugs. Mice were administered high doses of existing drug regimens for latent tuberculosis over a relatively short time period. Alanine aminotransferase (ALT), aspartate aminotransferase and bilirubin levels were determined after 2 weeks and 4 weeks of treatment in serum samples collected from uninfected mice as well as mice infected with Mycobacterium tuberculosis. ALT levels increased significantly after a RIF-PZA treatment regimen for 4 weeks in uninfected mice and after 2 weeks in infected mice. Bilirubin serum levels were also significantly elevated in the M. tuberculosis-infected mice after 4 weeks of RIF-PZA treatment. The data obtained indicate that changes in serum enzyme levels in mice after extensive exposure to tuberculosis drugs could be useful as an initial indicator of drug-related hepatotoxicity.
Asunto(s)
Alanina Transaminasa/metabolismo , Antituberculosos/administración & dosificación , Hígado/efectos de los fármacos , Pirazinamida/farmacología , Animales , Modelos Animales de Enfermedad , Quimioterapia Combinada , Hígado/enzimología , Pirazinamida/antagonistas & inhibidores , Pirazinamida/uso terapéutico , Rifampin/farmacología , Tuberculosis/tratamiento farmacológicoRESUMEN
This study extends earlier reports regarding the in vitro and in vivo efficacies of the nitroimidazopyran PA-824 against Mycobacterium tuberculosis. PA-824 was tested in vitro against a broad panel of multidrug-resistant clinical isolates and was found to be highly active against all isolates (MIC<1 microg/ml). The activity of PA-824 against M. tuberculosis was also assessed grown under conditions of oxygen depletion. PA-824 showed significant activity at 2, 10, and 50 microg/ml, similar to that of metronidazole, in a dose-dependent manner. In a short-course mouse infection model, the efficacy of PA-824 at 50, 100, and 300 mg/kg of body weight formulated in methylcellulose or cyclodextrin/lecithin after nine oral treatments was compared with those of isoniazid, rifampin, and moxifloxacin. PA-824 at 100 mg/kg in cyclodextrin/lecithin was as active as moxifloxacin at 100 mg/kg and isoniazid at 25 mg/kg and was slightly more active than rifampin at 20 mg/kg. Long-term treatment with PA-824 at 100 mg/kg in cyclodextrin/lecithin reduced the bacterial load below 500 CFU in the lungs and spleen. No significant differences in activity between PA-824 and the other single drug treatments tested (isoniazid at 25 mg/kg, rifampin at 10 mg/kg, gatifloxacin at 100 mg/kg, and moxifloxacin at 100 mg/kg) could be observed. In summary, its good activity in in vivo models, as well as its activity against multidrug-resistant M. tuberculosis and against M. tuberculosis isolates in a potentially latent state, makes PA-824 an attractive drug candidate for the therapy of tuberculosis. These data indicate that there is significant potential for effective oral delivery of PA-824 for the treatment of tuberculosis.
Asunto(s)
Antituberculosos , Mycobacterium tuberculosis/efectos de los fármacos , Nitroimidazoles , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Animales , Antituberculosos/administración & dosificación , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Recuento de Colonia Microbiana , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Pulmón/microbiología , Ratones , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/aislamiento & purificación , Nitroimidazoles/administración & dosificación , Nitroimidazoles/farmacología , Nitroimidazoles/uso terapéutico , Organismos Libres de Patógenos Específicos , Bazo/microbiología , Tuberculosis Pulmonar/tratamiento farmacológico , Tuberculosis Pulmonar/microbiologíaRESUMEN
The ability of Mycobacterium tuberculosis to grow in macrophages is central to its pathogenicity. We found previously that the widespread 210 strain of M. tuberculosis grew more rapidly than other strains in human macrophages. Because principal sigma factors influence virulence in some bacteria, we analysed mRNA expression of the principal sigma factor, sigA, in M. tuberculosis isolates during growth in human macrophages. Isolates of the 210 strain had higher sigA mRNA levels and higher intracellular growth rates, compared with other clinical strains and the laboratory strain H37Rv. SigA was also upregulated in the 210 isolate TB294 during growth in macrophages, compared with growth in broth. In contrast, H37Rv sigA mRNA levels did not change under these conditions. Overexpression of sigA enhanced growth of recombinant M. tuberculosis in macrophages and in lungs of mice after aerosol infection, whereas recombinant strains expressing antisense transcripts to sigA showed decreased growth in both models. In the presence of superoxide, sense sigA transformants showed greater resistance than vector controls, and the antisense sigA transformant did not grow. We conclude that M. tuberculosis sigA modulates the expression of genes that contribute to virulence, enhancing growth in human macrophages and during the early phases of pulmonary infection in vivo. This effect may be mediated in part by increased resistance to reactive oxygen intermediates.
Asunto(s)
Proteínas Bacterianas/química , Macrófagos/química , Mycobacterium tuberculosis/crecimiento & desarrollo , Fagocitos/microbiología , Factor sigma/fisiología , Animales , Proteínas Bacterianas/metabolismo , Células Cultivadas , Ácido Cítrico/farmacología , Regulación Bacteriana de la Expresión Génica , Humanos , Pulmón/microbiología , Pulmón/patología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Sondas de Oligonucleótidos , Polimorfismo de Longitud del Fragmento de Restricción , ARN Bacteriano/aislamiento & purificación , ARN Mensajero/química , ARN Mensajero/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor sigma/genética , Factor sigma/metabolismo , Tuberculosis/microbiología , Tuberculosis/patología , Vitamina K 3/farmacologíaRESUMEN
We have developed a rapid new in vivo method for screening experimental drugs for their activity against Mycobacterium tuberculosis by using the gamma interferon gene-disrupted (GKO) C57BL/6 mouse. Due to the rapid growth of the infection, statistical differences indicating positive efficacy of active compounds can be seen after only 8 days of treatment. To validate this model, several fluoroquinolones, including ciprofloxacin, levofloxacin, moxifloxacin, and gatifloxacin, were tested in parallel.
Asunto(s)
Antiinfecciosos/uso terapéutico , Tuberculosis/tratamiento farmacológico , Animales , Evaluación Preclínica de Medicamentos/métodos , Fluoroquinolonas , Interferón gamma , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosisRESUMEN
Previous studies examining the expression of adhesion and integrin molecules on CD4 T lymphocytes generated in response to virulent Mycobacterium tuberculosis infection revealed that certain inbred mouse strains susceptible to breakdown of chronic disease and subsequent reactivation had poor expression of these molecules, which might underlie their inability to adequately focus into lung tissues and mediate protection. The current study examines the possibility that prior vaccination with BCG, or a prior tuberculosis infection, would overcome this deficiency. It was found, however, that this was not the case. Whereas both resistant (C57BL/6) and susceptible (DBA/2, CBA/J) strains were equally well protected in the spleen after intravenous challenge, the latter strains were poorly protected in the lungs regardless of whether the challenge was given by the intravenous or aerosol route. Again, this was associated with poor up-regulation of adhesion and integrin molecules and with histological evidence in memory immune animals of a reduced and delayed influx of T lymphocytes into the lungs.