RESUMEN
Preliminary preclinical and observational studies suggest the potential utility of metformin as an adjunctive, host-directed agent for treatment of tuberculosis (TB). In this study, we sought to investigate the bactericidal and sterilizing activities of human-like exposures of metformin when given in combination with the first-line regimen against chronic tuberculosis in BALB/c mice. Mice receiving metformin adjunctive therapy had similar lung bacillary burdens with control mice during treatment, and the proportion of mice with microbiological relapse was similar between the two groups.
Asunto(s)
Antituberculosos/uso terapéutico , Carga Bacteriana/efectos de los fármacos , Isoniazida/uso terapéutico , Metformina/uso terapéutico , Mycobacterium tuberculosis/crecimiento & desarrollo , Pirazinamida/uso terapéutico , Rifampin/uso terapéutico , Tuberculosis Pulmonar/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Quimioterapia Combinada/métodos , Femenino , Pulmón/microbiología , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/efectos de los fármacos , RecurrenciaRESUMEN
BACKGROUND: The repurposing of existing agents may accelerate TB drug development. Recently, we reported that the lipid-lowering drug simvastatin, when added to the first-line antitubercular regimen, reduces the lung bacillary burden in chronically infected mice. OBJECTIVES: We investigated whether the addition of simvastatin to the first-line regimen (isoniazid/rifampicin/pyrazinamide) shortens the duration of curative TB treatment in mice. METHODS: Mycobacterium tuberculosis-infected THP-1 cells were exposed to simvastatin to determine the effect of statins on the activity of first-line anti-TB drug activity and intracellular rifampicin concentration. Single-dose and steady-state pharmacokinetic studies guided optimized simvastatin dosing in vivo. BALB/c mice were aerosol-infected with M. tuberculosis H37Rv and drug treatment was initiated 6 weeks post-infection. Separate groups of mice received standard TB treatment with or without simvastatin. Relapse rates were assessed 3 months after discontinuation of each treatment regimen. MALDI-MS imaging was used to image the cholesterol content of mouse lung lesions. RESULTS: Simvastatin significantly enhanced the bactericidal activity of first-line drugs against intracellular M. tuberculosis without altering intracellular rifampicin concentrations. Adjunctive treatment with 60 mg/kg simvastatin shortened the time required to achieve culture-negative lungs from 4.5 to 3.5 months. Following 2.5, 3.5 and 4.5 months of treatment, relapse rates were 100%, 50% and 0%, respectively, in the control group and 50% (Pâ=â0.03), 20% and 0%, respectively, in the statin group. Simvastatin did not alter plasma or lung lesion cholesterol levels. CONCLUSIONS: Statins are attractive candidates for host-directed, adjunctive TB therapy. Further preclinical studies are needed to define the optimal statin and dosing.
Asunto(s)
Antituberculosos/uso terapéutico , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis/tratamiento farmacológico , Animales , Línea Celular , Terapia Combinada/métodos , Modelos Animales de Enfermedad , Quimioterapia/métodos , Femenino , Humanos , Ratones Endogámicos BALB C , Monocitos/microbiología , Factores de Tiempo , Resultado del Tratamiento , Tuberculosis/microbiologíaRESUMEN
The repurposing of existing drugs is being pursued as a means by which to accelerate the development of novel regimens for the treatment of drug-susceptible and drug-resistant tuberculosis (TB). In the current study, we assessed the activity of the antipsychotic drug thioridazine (TRZ) in combination with the standard regimen in a well-validated murine TB model. Single-dose and steady-state pharmacokinetic studies were performed in BALB/c mice to establish human-equivalent doses of TRZ. To determine the bactericidal activity of TRZ against TB in BALB/c mice, three separate studies were performed, including a dose-ranging study of TRZ monotherapy and efficacy studies of human-equivalent doses of TRZ with and without isoniazid (INH) or rifampin (RIF). Therapeutic efficacy was assessed by the change in mycobacterial load in the lung. The human-equivalent dose of thioridazine was determined to be 25 mg/kg of body weight, which was well tolerated in mice. TRZ was found to accumulate at high concentrations in lung tissue relative to serum levels. We observed modest synergy during coadministration of TRZ with INH, and the addition of TRZ reduced the emergence of INH-resistant mutants in mouse lungs. In conclusion, this study further illustrates the opportunity to reevaluate the contribution of TRZ to the sterilizing activity of combination regimens to prevent the emergence of drug-resistant M. tuberculosis.
Asunto(s)
Antipsicóticos/uso terapéutico , Tioridazina/farmacocinética , Tioridazina/uso terapéutico , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Pulmonar/tratamiento farmacológico , Animales , Antipsicóticos/efectos adversos , Antipsicóticos/farmacocinética , Antituberculosos , Modelos Animales de Enfermedad , Reposicionamiento de Medicamentos , Farmacorresistencia Bacteriana Múltiple , Quimioterapia Combinada , Femenino , Isoniazida/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Rifampin/uso terapéutico , Tioridazina/efectos adversos , Tuberculosis Pulmonar/microbiologíaRESUMEN
We recently reported that in lung tissue, thioridazine accumulates at high concentrations relative to serum levels, displaying modest synergy with isoniazid and reducing the emergence of isoniazid-resistant mutants in mouse lungs. In this study, we sought to investigate the sterilizing activity of human-equivalent doses of thioridazine when given in combination with the "Denver regimen" against acute murine tuberculosis. We found a trend toward a positive impact of thioridazine on the bacterial clearance and lowering relapse rates of the combined standard TB chemotherapy.
Asunto(s)
Antituberculosos/uso terapéutico , Mycobacterium tuberculosis/efectos de los fármacos , Tioridazina/uso terapéutico , Tuberculosis Pulmonar/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Quimioterapia Combinada , Femenino , Isoniazida/uso terapéutico , Pulmón/microbiología , Ratones , Ratones Endogámicos BALB C , Pirazinamida/uso terapéutico , Rifampin/uso terapéuticoRESUMEN
BACKGROUND: The need to develop new, improved treatments for tuberculosis (TB) remains urgent, and the repurposing of existing drugs represents a possible shortcut to market. Recently, there has been significant interest in host-directed adjuvant therapy to enhance bacillary killing. HMG-CoA reductase inhibitors (statins), which are among the most commonly prescribed drugs, have immunomodulatory properties and improve the clinical outcomes of bacterial infections. METHODS: We studied the tuberculocidal activity of simvastatin alone and in combination with first-line anti-TB drugs in J774 macrophages and during chronic TB infection. RESULTS: Exposure to 5 µM simvastatin significantly increased the tuberculocidal activity of isoniazid in J774 macrophages at Day 3 after infection versus isoniazid alone (P=0.02). Similarly, relative to the standard oral regimen of rifampicin (10 mg/kg), isoniazid (10 mg/kg) and pyrazinamide (150 mg/kg) given five times weekly, the addition of 25 mg/kg simvastatin enhanced bacillary killing, reducing the number of lung cfu by an additional 1 log10 at Day 28 (P<0.01) and by a further 1.25 log10 at Day 56 (P<0.01). CONCLUSIONS: The potential additive activity of simvastatin to first-line TB treatment holds promise. However, further studies to identify the optimal statin and dosing are required. In addition the ability of combination treatment with statins to accelerate the time required to achieve a stable cure remains to be explored.
Asunto(s)
Antituberculosos/uso terapéutico , Sinergismo Farmacológico , Simvastatina/uso terapéutico , Tuberculosis/tratamiento farmacológico , Animales , Antituberculosos/farmacología , Carga Bacteriana , Línea Celular , Recuento de Colonia Microbiana , Modelos Animales de Enfermedad , Quimioterapia Combinada/métodos , Femenino , Isoniazida/farmacología , Isoniazida/uso terapéutico , Pulmón/microbiología , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Ratones Endogámicos BALB C , Pirazinamida/farmacología , Pirazinamida/uso terapéutico , Rifampin/farmacología , Rifampin/uso terapéutico , Simvastatina/farmacología , Resultado del TratamientoRESUMEN
Strategies involving new drug combinations, as well as new uses of existing drugs, are urgently needed to reduce the time required to cure patients with drug-sensitive or multidrug-resistant (MDR) tuberculosis (TB). We compared the sterilizing activity of the standard first-line antitubercular regimen, rifampin-isoniazid-pyrazinamide (RHZ), with that of the novel regimen PA-824-moxifloxacin-pyrazinamide (PaMZ), which is currently being studied in clinical trials (NCT01498419), in the guinea pig model of chronic TB infection, in which animals develop necrotic granulomas histologically resembling their human counterparts. Guinea pigs were aerosol infected with ~2 log10 bacilli of wild-type Mycobacterium tuberculosis H37Rv, and antibiotic treatment was initiated 6 weeks after infection. Separate groups of animals received RHZ, PaMZ, or single or two-drug components of the latter regimen administered at human-equivalent doses 5 days/week for a total of 8 weeks. Relapse rates were assessed 3 months after discontinuation of treatment to determine the sterilizing activity of each combination regimen. PaMZ given at human-equivalent doses was safe and well tolerated for the entire treatment period and rendered guinea pig lungs culture negative more rapidly than RHZ did. After 1 month of treatment, 80% and 50% of animals in the RHZ and PaMZ groups, respectively, had lung culture-positive relapse. Both combination regimens prevented microbiological relapse when administered for a total of 2 months. Our data support the use of PaMZ as a novel isoniazid- and rifamycin-sparing regimen suitable for treatment of both drug-sensitive TB and MDR-TB.
Asunto(s)
Antituberculosos/uso terapéutico , Nitroimidazoles/uso terapéutico , Pirazinamida/uso terapéutico , Rifamicinas/uso terapéutico , Tuberculosis Pulmonar/tratamiento farmacológico , Animales , Área Bajo la Curva , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Quimioterapia Combinada , Femenino , Cobayas , Pulmón/microbiología , Pulmón/patología , Mycobacterium tuberculosis , Nitroimidazoles/farmacocinética , Tamaño de los Órganos , Recurrencia , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológicoRESUMEN
OBJECTIVES: The antipsychotic drug thioridazine is active in the murine model of tuberculosis infection, which is predominantly intracellular in nature. Recent clinical reports suggest that thioridazine may play a role in the treatment of drug-resistant tuberculosis. We studied the tuberculocidal activity of thioridazine in guinea pigs, which develop necrotic lung granulomas histologically resembling their human counterparts. METHODS: Pharmacokinetic studies were performed in guinea pigs to establish human-equivalent doses of thioridazine. Guinea pigs were aerosol-infected with â¼100 bacilli of Mycobacterium tuberculosis and single-drug treatment was started 4 weeks later with a range of thioridazine doses daily (5 days/week) for up to 4 weeks. Control animals received no treatment or 60 mg/kg isoniazid. RESULTS: The human-equivalent dose of thioridazine was determined to be 5 mg/kg with saturable absorption noted above 50 mg/kg. At the start of treatment, the lung bacterial burden was â¼6.2 log10 cfu. Although isoniazid reduced bacillary counts more than 10-fold, thioridazine monotherapy showed limited killing over the range of doses tested, reducing lung bacillary counts by 0.3-0.5 log10 following 1 month of treatment. Thioridazine was tolerated up to 40 mg/kg. CONCLUSIONS: Thioridazine has limited bactericidal activity against extracellular bacilli within necrotic granulomas. Its contribution to the sterilizing activity of combination regimens against drug-susceptible and drug-resistant tuberculosis remains to be determined.
Asunto(s)
Antipsicóticos/uso terapéutico , Tioridazina/uso terapéutico , Tuberculosis/tratamiento farmacológico , Aerosoles , Animales , Antipsicóticos/efectos adversos , Antipsicóticos/farmacocinética , Área Bajo la Curva , Relación Dosis-Respuesta a Droga , Determinación de Punto Final , Espacio Extracelular , Femenino , Cobayas , Humanos , Ratones , Mycobacterium tuberculosis/efectos de los fármacos , Tamaño de los Órganos/efectos de los fármacos , Tioridazina/efectos adversos , Tioridazina/farmacocinética , Tuberculosis/microbiología , Tuberculosis/patologíaRESUMEN
Rifamycins are key sterilizing drugs in the current treatment of active tuberculosis (TB). Daily dosing of rifapentine (P), a potent rifamycin with high intracellular accumulation, in place of rifampin (R) in the standard antitubercular regimen significantly shortens the duration of treatment needed to prevent relapse in a murine model of active TB. We undertook the current study to compare directly the activities of human-equivalent doses of P and R in a guinea pig model of chronic TB, in which bacilli are predominantly extracellular within human-like necrotic granulomas. Hartley strain guinea pigs were aerosol infected with ~200 bacilli of Mycobacterium tuberculosis H37Rv, and treatment given 5 days/week was initiated 6 weeks later. R at 100 mg/kg of body weight and P at 100 mg/kg were given orally alone or in combination with isoniazid (H) at 60 mg/kg and pyrazinamide (Z) at 300 mg/kg. Culture-positive relapse was assessed in subgroups of guinea pigs after completion of 1 and 2 months of treatment. Human-equivalent doses of R and P showed equivalent bactericidal activity when used alone and in combination therapy. In guinea pigs treated with rifampin, isoniazid, and pyrazinamide (RHZ) or PHZ, microbiological relapse occurred in the lungs of 8/10 animals treated for 1 month and in 0/10 animals treated for 2 months. Substitution of P for R in the standard antitubercular regimen did not shorten the time to cure in this guinea pig model of chronic TB. Data from ongoing clinical trials comparing the activity of these two drugs are awaited to determine the relevance of the guinea pig TB model in preclinical drug screening.
Asunto(s)
Antituberculosos/uso terapéutico , Rifampin/análogos & derivados , Rifampin/uso terapéutico , Tuberculosis/tratamiento farmacológico , Animales , Femenino , Cobayas , Isoniazida/uso terapéutico , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/patogenicidad , Pirazinamida/uso terapéuticoRESUMEN
OBJECTIVES: It is widely believed that persistent Mycobacterium tuberculosis inhabits necrotic lung granulomas in humans and that the microenvironmental conditions encountered therein render the bacilli phenotypically tolerant to antibiotics, accounting for the long duration required for successful treatment of tuberculosis (TB). To validate this belief, we directly compared the activity of rifampicin/isoniazid/pyrazinamide (RHZ) against chronic TB infection in guinea pigs, which exhibit caseous granulomas histologically resembling human caseous foci, and in mice, which lack necrotic granulomas. METHODS: Guinea pigs and mice were aerosol-infected with M. tuberculosis CDC1551 and twice weekly treatment with RHZ was started 4 weeks later. Culture-positive relapse was assessed in subgroups of guinea pigs after 3 months and 4 months of treatment. RESULTS: All guinea pig lungs exhibited histological evidence of granulomas with central caseation, while mouse lungs exhibited cellular lesions at the initiation of antibiotic treatment. Guinea pig lungs became culture-negative after 2 months of RHZ given twice weekly at human-equivalent doses. Relapse rates in guinea pigs were 0% (0/10) both after 3 months and 4 months of treatment. In contrast, all mouse lungs remained culture-positive after 4 months of equivalent RHZ exposures. CONCLUSIONS: Caseous necrosis does not reduce the sterilizing activity of the standard antituberculosis regimen of RHZ. Our findings have important implications for the use of alternative animal models in testing novel TB drug regimens and for modelling M. tuberculosis persistence.
Asunto(s)
Antituberculosos/administración & dosificación , Farmacorresistencia Bacteriana , Quimioterapia/métodos , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis/tratamiento farmacológico , Animales , Antituberculosos/farmacología , Modelos Animales de Enfermedad , Femenino , Cobayas , Ratones , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/aislamiento & purificación , Recurrencia , Enfermedades de los Roedores/tratamiento farmacológico , Enfermedades de los Roedores/microbiología , Enfermedades de los Roedores/patología , Tuberculosis/microbiología , Tuberculosis/patologíaRESUMEN
A long duration of treatment and emerging drug resistance pose significant challenges for global tuberculosis (TB) eradication efforts. Therefore, there is an urgent need to develop novel strategies to shorten TB treatment regimens and to treat drug-resistant TB. Using an albumin-fusion strategy, we created a novel albumin-fused granulocyte-macrophage colony-stimulating factor (albGM-CSF) molecule that harnesses albumin's long half-life and targeting abilities to enhance the biostability of GM-CSF and direct it to the lymph nodes, where the effects of GM-CSF can increase dendritic cell populations crucial for eliciting a potent immune response. In this study, we demonstrate that albGM-CSF serves as a novel immunotherapy for chronic Mycobacterium tuberculosis (Mtb) infections by enhancing GM-CSF biostability in serum. Specifically, albumin is very safe, stable, and has a long half-life, thereby enhancing the biostability of GM-CSF. In the lungs and draining lymph nodes, albGM-CSF is able to increase the numbers of dendritic cells, which are crucial for the activation of naive T cells and for eliciting potent immune responses. Subcutaneous administration of albGM-CSF alone reduced the mean lung bacillary burden in mice with chronic tuberculosis infection. While GM-CSF administration was associated with IL-1ß release from Mtb-infected dendritic cells and macrophages, higher IL-1ß levels were observed in albGM-CSF-treated mice with chronic tuberculosis infection than in mice receiving GM-CSF. Albumin fusion with GM-CSF represents a promising strategy for the control of chronic lung tuberculosis infections and serves as a novel therapeutic vaccination platform for other infectious diseases and malignancies.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Albúminas/farmacología , Animales , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Inmunoterapia , Ratones , Tuberculosis/terapiaRESUMEN
OBJECTIVES: The biphasic kill curve of isoniazid against Mycobacterium tuberculosis in guinea pigs is due to the presence of persisters rather than selection of isoniazid-resistant mutants. To determine whether this phenomenon is common to other bactericidal drugs, we studied the activity of streptomycin and its ability to select for streptomycin-resistant mutants in the guinea pig model of tuberculosis. METHODS: Pharmacokinetic studies were performed to establish the human-equivalent dose of streptomycin. Guinea pigs were aerosol-infected with M. tuberculosis and 2 weeks later streptomycin was given for 5 days/week via intramuscular injection. Bactericidal activity was assessed by homogenizing and plating lungs for cfu until 10 weeks of treatment. At each timepoint, cfu were isolated, suspended in normal saline and re-plated on plates containing 0.5, 1.0, 2.0 or 10.0 mg/L streptomycin. RESULTS: The human-equivalent dose of streptomycin was determined to be 70 mg/kg. Streptomycin showed potent activity during the first 14 days of treatment, rescuing all animals from acute tuberculosis-related death and reducing lung cfu by â¼4 log(10). However, streptomycin activity was dramatically reduced thereafter, as lung cfu declined by only â¼1 log(10) over the next 56 days of treatment. Although streptomycin-resistant mutants were detectable, their frequency of isolation was identical at treatment initiation and after 70 days of treatment. CONCLUSIONS: The reduced activity of streptomycin during the second phase of monotherapy is not associated with the selection of streptomycin-resistant mutants but, rather, with the presence of phenotypically tolerant 'persisters'.
Asunto(s)
Antibacterianos/farmacología , Viabilidad Microbiana/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Estreptomicina/farmacología , Tuberculosis/tratamiento farmacológico , Animales , Antibacterianos/administración & dosificación , Recuento de Colonia Microbiana , Modelos Animales de Enfermedad , Farmacorresistencia Bacteriana , Tolerancia a Medicamentos , Femenino , Cobayas , Inyecciones Intramusculares , Pulmón/microbiología , Estreptomicina/administración & dosificaciónRESUMEN
OBJECTIVES: In this study, we sought to compare the sterilizing activity of human-equivalent doses of the 'Denver regimen' against acute tuberculosis (TB) infection in the standard mouse model and in the guinea pig. METHODS: Pharmacokinetic studies in guinea pigs were used to establish human-equivalent doses for rifampicin, isoniazid and pyrazinamide. Guinea pigs and mice were aerosol-infected with Mycobacterium tuberculosis CDC1551 and treatment was started 2 weeks later with rifampicin/isoniazid/pyrazinamide for up to 6 months. For the first 2 weeks of therapy, the dosing frequency was 5 days/week, and for the remaining period, twice weekly. Treatment was discontinued in groups of 30 mice and 10 guinea pigs at 5 months and at 6 months, and these animals were held for a further 3 months in order to assess relapse rates. RESULTS: Guinea pig lungs became culture-negative after 3 months of predominantly twice-weekly treatment and relapse rates were 0% (0/10) both after 5 months and after 6 months of treatment. In contrast, all mice remained culture-positive despite 6 months of the same treatment, and 93% (28/30) and 69% (20/29) of mice relapsed after treatment for 5 and 6 months, respectively. CONCLUSIONS: Treatment with rifampicin/isoniazid/pyrazinamide administered at human-equivalent doses is much more potent against acute TB infection in guinea pigs than in mice. Our findings have important implications for the use of alternative animal models in testing novel TB drug regimens and for modelling M. tuberculosis persistence.
Asunto(s)
Antituberculosos/uso terapéutico , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis/tratamiento farmacológico , Animales , Antituberculosos/administración & dosificación , Antituberculosos/farmacocinética , Modelos Animales de Enfermedad , Femenino , Cobayas , Isoniazida/administración & dosificación , Isoniazida/farmacocinética , Isoniazida/uso terapéutico , Pulmón/microbiología , Ratones , Ratones Endogámicos BALB C , Pirazinamida/administración & dosificación , Pirazinamida/farmacocinética , Pirazinamida/uso terapéutico , Rifampin/administración & dosificación , Rifampin/farmacocinética , Rifampin/uso terapéutico , Resultado del TratamientoRESUMEN
The lengthy and complicated current regimen required to treat drug-susceptible tuberculosis (TB) reflects the ability of Mycobacterium tuberculosis (Mtb) to persist in host tissues. The stringent response pathway, governed by the dual (p)ppGpp synthetase/hydrolase, Rel Mtb , is a major mechanism underlying Mtb persistence and antibiotic tolerance. In the current study, we addressed the hypothesis that Rel Mtb is a "persistence antigen" presented during TB chemotherapy and that enhanced immunity to Rel Mtb can enhance the tuberculocidal activity of the first-line anti-TB drug, isoniazid, which has reduced efficacy against Mtb persisters. C57BL/6 mice and Hartley guinea pigs were aerosol-infected with M. tuberculosis (Mtb) and, 4 weeks later, received either human-equivalent daily doses of isoniazid alone, or isoniazid in combination with a DNA vaccine targeting relMtb . After isoniazid treatment, there was a significant reduction in dominant antigen ESAT6-reactive CD4+ or TB10.4-reactive CD8+ T cells in the lungs and spleens of mice. However, the total number of Rel Mtb -reactive CD4+ T cells remained stable in mouse lungs and spleens, as did the number of Rel Mtb -reactive CD8+T cells. Therapeutic vaccination with relMtb DNA vaccine enhanced the activity of isoniazid in Mtb-infected C57BL/6 mice and guinea pigs. When treatment with isoniazid was discontinued, mice immunized with the relMtb DNA vaccine showed a lower mean lung bacterial burden at relapse compared to the control group. Our work shows that antitubercular treatment shapes the antigenic environment, and that therapeutic vaccination targeting the Mtb stringent response may represent a novel approach to enhance immunity against Mtb persisters, with the ultimate goal of shortening curative TB treatment.
Asunto(s)
Antituberculosos/uso terapéutico , Isoniazida/uso terapéutico , Mycobacterium tuberculosis/inmunología , Vacunas contra la Tuberculosis/uso terapéutico , Tuberculosis/tratamiento farmacológico , Tuberculosis/prevención & control , Vacunación/métodos , Vacunas de ADN/uso terapéutico , Animales , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular , Enfermedad Crónica/tratamiento farmacológico , Enfermedad Crónica/prevención & control , Farmacorresistencia Bacteriana/inmunología , Femenino , Guanosina Pentafosfato/metabolismo , Cobayas , Hidrolasas/inmunología , Hidrolasas/metabolismo , Ligasas/inmunología , Ligasas/metabolismo , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/enzimología , Resultado del Tratamiento , Tuberculosis/inmunología , Tuberculosis/microbiología , Vacunas de ADN/inmunologíaRESUMEN
Fatty acid synthase (FAS), the enzyme responsible for the de novo synthesis of fatty acids, is highly expressed in ovarian cancers and most common human carcinomas. Inhibition of FAS and activation of AMP-activated protein kinase (AMPK) have been shown to be cytotoxic to human cancer cells in vitro and in vivo. In this report, we explore the cytotoxic mechanism of action of FAS inhibition and show that C93, a synthetic FAS inhibitor, increases the AMP/ATP ratio, activating AMPK in SKOV3 human ovarian cancer cells, which leads to cytotoxicity. As a physiologic consequence of AMPK activation, acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid synthesis, was phosphorylated and inhibited whereas glucose oxidation was increased. Despite these attempts to conserve energy, the AMP/ATP ratio increased with worsening cellular redox status. Pretreatment of SKOV3 cells with compound C, an AMPK inhibitor, substantially rescued the cells from C93 cytotoxicity, indicating its dependence on AMPK activation. 5-(Tetradecyloxy)-2-furoic acid, an ACC inhibitor, did not activate AMPK despite inhibiting fatty acid synthesis pathway activity and was not significantly cytotoxic to SKOV3 cells. This indicates that substrate accumulation from FAS inhibition triggering AMPK activation, not end-product depletion of fatty acids, is likely responsible for AMPK activation. C93 also exhibited significant antitumor activity and apoptosis against SKOV3 xenografts in athymic mice without significant weight loss or cytotoxicity to proliferating cellular compartments such as bone marrow, gastrointestinal tract, or skin. Thus, pharmacologic FAS inhibition selectively activates AMPK in ovarian cancer cells, inducing cytotoxicity while sparing most normal human tissues from the pleiotropic effects of AMPK activation.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Ácido Graso Sintasas/antagonistas & inhibidores , Complejos Multienzimáticos/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Activación Enzimática , Ácidos Grasos/metabolismo , Femenino , Furanos/farmacología , Glucosa/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , NAD/metabolismo , Neoplasias Ováricas/metabolismo , Oxidación-Reducción , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mycobacterium tuberculosis (Mtb) remains a global health challenge due to the limited efficacy of the Mtb vaccine in current use, Bacillus Calmette-Guérin (BCG). To date, there is no available vaccine for immunocompromised individuals. Thus, there is an urgent need to develop a new vaccine candidate which can induce mucosal immunity in hosts with different immune statuses. DnaK (HSP70) has been shown to induce protective immunity against Mtb infection when administered by DNA vaccine; however, the protection is inferior to that induced by the BCG vaccine. In our study, we vaccinated C57BL/6J mice with DnaK protein alone. Subcutaneous or intranasal vaccination with DnaK generated IFNγ-secreting CD4+ T cells in the spleen, but only intranasal vaccination generated IL-17-releasing CD4+ T cells in the lungs, even when circulating CD4+ T cells were diminished. Furthermore, intranasal vaccination with DnaK generated tissue resident CD4+ T cells in the lungs. Vaccination with DnaK alone resulted in protective immunity comparable to BCG vaccination against tuberculosis in mice. Our results demonstrate that intranasal vaccination with DnaK can generate mucosal immunity in immunocompromised or immunocompetent mice and DnaK vaccination can generate protection against Mtb similar to BCG, underscoring its potential utility as an Mtb vaccine candidate in humans.
Asunto(s)
Adenosina Trifosfatasas/inmunología , Proteínas Bacterianas/inmunología , Linfocitos T CD4-Positivos/inmunología , Inmunidad Mucosa , Chaperonas Moleculares/inmunología , Mycobacterium tuberculosis/inmunología , Mucosa Nasal/inmunología , Mucosa Nasal/microbiología , Tuberculosis/inmunología , Administración Intranasal , Animales , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Vacuna BCG/inmunología , Reacciones Cruzadas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Inmunización , Depleción Linfocítica , Ratones , Vacunas contra la Tuberculosis/administración & dosificación , Vacunas contra la Tuberculosis/inmunologíaRESUMEN
High levels of fatty acid synthase (FAS) have been found in cancer precursor lesions of the colon, stomach, esophagus, oral cavity, prostate, and breast. Inhibition of FAS with C75 has led to a significant antitumor effect in both human breast and prostate cancer xenografts. Recently, HER2/neu, which has also been identified in preneoplastic breast lesions, has been shown to regulate FAS expression through the PI3K/Akt signal transduction pathway rendering them susceptible to FAS inhibition. Utilizing the neu-N transgenic mouse model of mammary cancer, weekly treatment of the neu-N mice with C75 (30 mg/kg) for 10 weeks significantly delayed tumor progression. Only 20% of the C75-treated transgenic mice developed mammary carcinoma by 220 days, compared to 50% in the vehicle control animals. Two C75-treated animals never developed mammary cancer. Analysis of mammary tissue following 10 weeks of C75 treatment revealed a significant delay in mammary maturation as manifested by a reduction of the number and caliber of mammary ducts and budding epithelial structures. Apoptotic changes were increased, DNA synthesis was decreased, and the expressions of FAS, neu, Akt, phospho-Akt, and p21(waf1) were all decreased when compared to vehicle controls and FVB/N mice. Importantly, these effects were restricted to the breast epithelial cells that overexpressed neu, not involving other normal duct structures in the skin, liver, or kidney. C247, an FAS inhibitor chemically distinct from C75, significantly delayed mammary maturation similar to C75. Thus, pharmacological inhibition of FAS affects the expression of key oncogenes involved in both cancer development and maintenance of the malignant phenotype. Moreover, these data identify FAS as a potential novel drug target for breast cancer chemoprevention.
Asunto(s)
4-Butirolactona/análogos & derivados , Ácido Graso Sintasas/antagonistas & inhibidores , Neoplasias Mamarias Animales/prevención & control , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , 4-Butirolactona/farmacología , Animales , Proteínas de Unión al ADN , Femenino , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/crecimiento & desarrollo , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Células Tumorales Cultivadas , Receptor fasRESUMEN
C75, an inhibitor of fatty acid synthase (FAS), induces apoptosis in cultured human cancer cells. Its proposed mechanism of action linked high levels of malonyl-CoA after FAS inhibition to potential downstream effects including inhibition of carnitine palmitoyltransferase-1 (CPT-1) with resultant inhibition of fatty acid oxidation. Recent data has shown that C75 directly stimulates CPT-1 increasing fatty acid oxidation in MCF-7 human breast cancer cells despite inhibitory concentrations of malonyl-CoA. In light of these findings, we have studied fatty acid metabolism in MCF7 human breast cancer cells to elucidate the mechanism of action of C75. We now report that: (a) in the setting of increased fatty acid oxidation, C75 inhibits fatty acid synthesis; (b) C273, a reduced form of C75, is unable to inhibit fatty acid synthesis and is nontoxic to MCF7 cells; (c) C75 and 5-(tetradecyloxy)-2-furoic acid (TOFA), an inhibitor of acetyl-CoA carboxylase, both cause a significant reduction of fatty acid incorporation into phosphatidylcholine, the major membrane phospholipid, within 2 h; (d) pulse chase studies with [(14)C]acetate labeling of membrane lipids show that both C75 and TOFA accelerate the decay of (14)C-labeled lipid from membranes within 2 h; (e) C75 also promotes a 2-3-fold increase in oxidation of membrane lipids within 2 h; and (f) because interference with phospholipid synthesis during S phase is known to trigger apoptosis in cycling cells, we performed double-labeled terminal deoxynucleotidyltransferase-mediated nick end labeling and BrdUrd analysis with both TOFA and C75. C75 triggered apoptosis during S phase, whereas TOFA did not. Moreover, application of TOFA 2 h before C75 blocked the C75 induced apoptosis, whereas etomoxir did not. Taken together these data indicate that FAS inhibition and its downstream inhibition of phospholipid production is a necessary part of the mechanism of action of C75. CPT-1 stimulation does not likely play a role in the cytotoxic response. The continued ability of TOFA to rescue cancer cells from C75 cytotoxicity implies a proapoptotic role for malonyl-CoA independent of CPT-1 that selectively targets cancer cells as they progress into S phase.
Asunto(s)
4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacología , Apoptosis/efectos de los fármacos , Ácido Graso Sintasas/antagonistas & inhibidores , Furanos/farmacología , Apoptosis/fisiología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Carnitina O-Palmitoiltransferasa/metabolismo , División Celular/efectos de los fármacos , Línea Celular Tumoral , Interacciones Farmacológicas , Compuestos Epoxi/farmacología , Humanos , Lípidos de la Membrana/biosíntesis , Lípidos de la Membrana/metabolismo , Fosfolípidos/biosíntesis , Fase S/efectos de los fármacos , Fase S/fisiologíaRESUMEN
Novel drugs are required to shorten the duration of treatment for tuberculosis (TB) and to combat the emergence of drug resistance. One approach has been to identify and target Mycobacterium tuberculosis (Mtb) virulence factors, which promote the establishment of TB infection and pathogenesis. Mtb produces a number of virulence factors, including two protein tyrosine phosphatases (PTPs), mPTPA and mPTPB, to evade the antimicrobial functions of host macrophages. To assess the therapeutic potential of targeting the virulent Mtb PTPs, we developed highly potent and selective inhibitors of mPTPA (L335-M34) and mPTPB (L01-Z08) with drug-like properties. We tested the bactericidal activity of L335-M34 and L01-Z08 alone or together in combination with the standard antitubercular regimen of isoniazid-rifampicin-pyrazinamide (HRZ) in the guinea pig model of chronic TB infection, which faithfully recapitulates some of the key histological features of human TB lesions. Following a single dose of L335-M34 50mg/kg and L01-Z08 20 mg/kg, plasma levels were maintained at levels 10-fold greater than the biochemical IC50 for 12-24 hours. Although neither PTP inhibitor alone significantly enhanced the antibacterial activity of HRZ, dual inhibition of mPTPA and mPTPB in combination with HRZ showed modest synergy, even after 2 weeks of treatment. After 6 weeks of treatment, the degree of lung inflammation correlated with the bactericidal activity of each drug regimen. This study highlights the potential utility of targeting Mtb virulence factors, and specifically the Mtb PTPs, as a strategy for enhancing the activity of standard anti-TB treatment.
RESUMEN
C75, a synthetic inhibitor of fatty acid synthase (FAS), causes anorexia and profound weight loss in lean and genetically obese mice. C75 also acts as a stimulator of carnitine palmitoyltransferase-1 to induce fatty acid oxidation. To approximate human obesity, we used a 2-wk C75 treatment model for diet-induced obese (DIO) mice to investigate the central and peripheral effects of C75 on gene expression. C75 treatment decreased food intake, increased energy expenditure, and reduced body weight more effectively in DIO than in lean mice. Analysis of the gene expression changes in hypothalamus demonstrated that the reduced food intake in C75-treated DIO mice might be mediated by inhibition of orexigenic neuropeptide expression and induction of anorexigenic neuropeptide expression. Gene expression changes in peripheral tissues indicated that C75 increased energy expenditure by the induction of genes involved in fatty acid oxidation. C75 also inhibited the expression of genes in peripheral tissues responsible for fatty acid synthesis and accumulation. The patterns of the changes in central and peripheral gene expression that occur with C75 treatment provide mechanisms to explain the reduced food intake and increased energy expenditure observed with C75.