1 Tuberculosis Nuevos Tratamientos en Tuberculosis MultidrogoresistenteDr Eduardo Sada Diaz Instituto Nacional de Enfermedades Respiratorias Junio 2015

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3 TUBERCULOSIS ANTECEDENTESEn 2012 hubo 8.7 millones de nuevos casos de tuberculosis activa. Los avances en el diagnostico , farmacos y vacunas asi como intervenciones han permitido mantener optimismo en el futuro control de la tuberculosis OMS 2013

4 INCIDENCIA GLOBAL EN TUBERCULOSISFigure 1 Global Incidence of Tuberculosis. Panel A shows global trends in the estimated incidence of tuberculosis from 1990 through 2011 among all patients, those with human immunodeficiency virus (HIV) coinfection, and without HIV coinfection. The shading around the data curves indicates uncertainty intervals on the basis of available data. Panel B shows the estimated global incidence of tuberculosis in 2011. Zumla A et al. N Engl J Med 2013;368:

5 NUMEROS GLOBALES DE CASOS DE TUBERCULOSIS MULTIDROGORESISTENTEFigure 2 Global Numbers of Cases of Multidrug-Resistant Tuberculosis. Shown are the estimated numbers of cases of multidrug-resistant disease (including extensively drug-resistant disease) among cases of pulmonary tuberculosis that were officially reported in 2011. Zumla A et al. N Engl J Med 2013;368:

6 Incidencia estimada 2012 México SSA18,999 casos nuevos TB 81.4% pulmonar (15,457) 152 casos nuevos de TB MDR 20.9% TB/DM 7.4% TB/SIDA TASA ( habs.) >24 <7.10 Plataforma Única de Información /SUIVE/DGE/SS. Cierre 2012

7 Tuberculosis Farmaco-resistente (Casos nuevos 2000 – 2010)

8 PAPEL DE LA CLINICA DE TUBERCULOSIS EN EL INER

9 Problemas Actuales PrioridadesCoinfecciòn TB –HIV Tratamientos Ineficientes con Aparición de cepas MDR Y XDR Poblaciòn Terapia Actual Prioridades TB drogosensible 4 drogas > 6 Meses 2RHZE + 4 RH Regimenes cortos Terapias Simples TB Resistente MDR XDR Drogas Secundarias Inyectables 48 meses Tratamiento Oral Corto -Eficaz -Seguro TB HIV Interacciòn drogas TB-HIV No interacciòn Coadminitracion segura TB latentes Isoniazida 6-9 meses Terapia Corta –Segura

10 INICIAL 1 AÑO DESPUES Es importante tambien tener en cuenta el costo del paciente y o me refiere a costo economico, si no a costo en cuento funcionalidad pulmonar

11 TBP MDR Diseñar esquema de Tx Audiología Psiquiatría Cirugía ClínicoCultivo y PFS Historial farmacológico Abasto de fármacos Diseñar esquema de Tx Numero de fármacos a recibir Efectos adversos Probabilidades de curación CONSENTIMIENTO HOSPITALIZACION Laboratorios Audiología Psiquiatría Cirugía Nefrología Sd. Metabólico TX ESCALONADO 20 PACIENTES (46%) TENIAN USO PREVIO DE AMINOGLUCOSIDO (17 S Y 3 OTROS AMINOGLUCOSIDOS) (2 PACTES RECIBIERON 2 AMINOGLUCOSIDOS DIFERENTES) NEFROLOGIA: EN PROMEDIO 6 MESES DE AMINOGLUCOSIDO IV MINIMO 130 DOSIS MAXIMO 225 5 PACIENTES YA TENIAN DAÑO RENAL CRONICO CON IN IFG <60ML/MINT 8 PACIENTES TENIAN HIPERFILTRACION GLOMERUALR IFG >120ML/MINT Valorar tolerancia Monitorizar efectos adversos y reacciones alérgicas Preparar al personal de salud Corroborar el abastecimiento de fármacos CENTRO DE SALUD Clínico Microbiológico Radiológico

12 Programa de TBP farmaco-resistente en el INERPeríodo 2010 – 2012 Edad Promedio 44años 43 MDR Relaciòn H/M 1 XDR Promedio de edad: años Promedio de edad: años

13 A 9-month regimen for MDR-TB in BangladeshGatifloxacina Etambutol Pyrazinamida Clofazimina Kanamycina Protionamida Isoniazida 4-meses fase intensiva y prolongable si el cultivo seguia + al 4 mes. Fase de continuacion 5 meses AJRCCM 2010:182:684-92

14 Perspectivas Históricas1940 Tratamiento paliativo ( Inmunoterapia- Ambiental -Nutricional )Mortalidad 50% 1944 Introducción de Estreptomicina y PAS 1952 Introducción de Isoniazida 1970 Regimenes combinados 18 meses Regimenes Acortados RHZE (BMC ) 2RHZE Desarrollo de Nuevas drogas para TB MDR /

15 Desarrollo Pre-ClínicoGLP Toxicologia CPZEN-45 FASE I DC-159a Q-201 SPR-10199 SQ-609 SQ-641 BTZ043 TBA-354 FASE II FASE III AZD5847 Oxazolidinona BEDAQUILINE (TMC207) TB-MDR y TB-DS Diarylquinolina Linezolid para MDR-TB Nuevos Regímenes Nitroimidazol-oxazina, diarylquinolina, FQ,Derivados del Ac. Nicotínico RIFAPENTINA (DS-TB) SQ-109 Etilenediamina PA-824 Nitroimidazol-oxazina GATIFLOXACINO para DS-TB MOXIFLOXACINO TB latente DELAMANID (OPC67683) TB MDR Nitro-dihidro-imidazooxazole Desarrollo Pre-Clínico Desarrollo Clínico Quinolonas Oxazolidinona Nitroimidazoles Rifampicinas Sutezolid (PNU ) Oxazolidinona

16 Mecanismo de Acción Bio- reduction DNA Reactive Species mRNA H+ ADPPeptide H+ ADP ATP Bio- reduction Diferentes dianas PA-824 OPC (Delamanid) ADN Gyrasa Gatifloxacin Moxifloxacin ARN Polymerasa Rifapentina Ribosoma PNU (Sutezolid) AZD-5847 ATP Synthase TMC-207 (bedaquiline) Pared celular SQ-109

17 Original Article Delamanid for Multidrug-Resistant Pulmonary TuberculosisN Engl J Med Volume 366(23): June 7, 2012

18 Conclusiones Delamanid se asocio con un incremento en la conversion a dos meses en pacientes con TB MDR. Este resultado demuestra que Delamanid puede ser una opciòn para el tratamiento de pacientes con TB MDR .

19 . Original Article The Diarylquinoline TMC207 for Multidrug-Resistant Tuberculosis N Engl J Med Volume 360(23): June 4, 2009 TMC Bedaquilina

20 Conclusion La actividad clinica de la droga TMC207 demuestra que las ATP sintetasas de MTB MDR son susceptibles de ser afectados por este grupo de medicamentos y por lo tanto son drogas utiles para el tratamiento de TB MDR.

21 Original Article Linezolid for Treatment of Chronic Extensively Drug-Resistant TuberculosisMyungsun Lee, M.D., Jongseok Lee, Ph.D., Matthew W. Carroll, M.D., Hongjo Choi, M.D., Seonyeong Min, R.N., Taeksun Song, Ph.D., Laura E. Via, Ph.D., Lisa C. Goldfeder, C.C.R.P., Eunhwa Kang, M.Sc., Boyoung Jin, R.N., Hyeeun Park, R.N., Hyunkyung Kwak, B.S., Hyunchul Kim, Ph.D., Han-Seung Jeon, M.S., Ina Jeong, M.D., Joon Sung Joh, M.D., Ray Y. Chen, M.D., Kenneth N. Olivier, M.D., Pamela A. Shaw, Ph.D., Dean Follmann, Ph.D., Sun Dae Song, M.D., Ph.D., Jong-Koo Lee, M.D., Dukhyoung Lee, M.D., Cheon Tae Kim, M.D., Veronique Dartois, Ph.D., Seung-Kyu Park, M.D., Sang-Nae Cho, D.V.M., Ph.D., and Clifton E. Barry, III, Ph.D. N Engl J Med Volume 367(16): October 18, 2012

22 Justificación Es necesario buscar nuevas alternativas terapéuticasIncidencia: 9 x 106 casos 1.5 millones de muertes1 TB Latente La eficacia de la vacuna BCG es variable2 El régimen de tratamiento de 6 meses La Isoniazida como terapia preventiva3 Emergencia de cepas drogo-resistentes Justificación Es necesario buscar nuevas alternativas terapéuticas Instead, such responses cause M. tuberculosis to adopt a clinically silent, latent state of infection, from which the bacteria can be reactivated. (1/3 de la población) As a practical problem, the limited understanding of immunity to M. tuberculosis deters rapid progress in developing TB vaccines that are superior to Mycobacterium bovis (BCG), a vaccine developed in the early twentieth century that has limited efficacy in preventing active tuberculosis, despite having been administered to >3.5 billion people WHO, Fine PE, The Lancet WHO, Treatment Guidelines, 2010

23 AUTOFAGIA. Choi AM et al. N Engl J Med 2013;368:651-662.Figure 1. Phases of the Autophagic Pathway. The autophagic pathway proceeds through several phases, including initiation (formation of a preautophagosomal structure leading to an isolation membrane, or phagophore), vesicle elongation, autophagosome maturation and cargo sequestration, and autophagosome–lysosome fusion. In the final stage, autophagosomal contents are degraded by lysosomal acid hydrolases and the contents of the autolysosome are released for metabolic recycling. Choi AM et al. N Engl J Med 2013;368:

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25 El tratamiento con fármacos favorece elEl tratamiento con fármacos favorece el reclutamiento de Mtb dentro de Autofagosomas MTB HOESCHT LC3 MTB MTB-LC3+ MTB+L MTB+C MTB+R

26 Conclusiòn La TB MDR constituye un problema de salud o publica con un alto costo para los pacientes y para las instituciones de salud. Existen nuevas drogas para el tratamiento de TB MDR lo que establece la posibilidad de una mejor manejo terapeutico de estos pacientes. Se requieren nuevas ideas y alternativas terapeuticas en el tratamiento de Tuberculosis,.

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28 Figure 8. Morphology of macrophage cell death in pulmonary TB.Repasy T, Lee J, Marino S, Martinez N, et al. (2013) Intracellular Bacillary Burden Reflects a Burst Size for Mycobacterium tuberculosis In Vivo. PLoS Pathog 9(2): e doi: /journal.ppat

29 Effects of Autophagy on Disease Progression.Figure 3. Effects of Autophagy on Disease Progression. Autophagy influences a number of processes that have various effects on disease progression. Processes that are beneficial (i.e., that inhibit disease progression) are shown in blue, and those that are putatively detrimental (i.e., that promote disease progression) are shown in red. In many diseases, autophagy plays a common role that involves clearance of dysfunctional mitochondria and protein aggregates. The disease mechanisms discussed in this review may be related to the failure of autophagy to perform these functions. Additional mechanisms may include a role for autophagy in the regulation of cell death and proliferation, as shown in models of cardiac and lung diseases. In cancer, autophagy may prevent tumorigenesis but may also promote tumor-cell survival and tumor growth, thus variably affecting the efficacy of anticancer therapies. In infectious disease, autophagy plays a direct role in clearing intracellular pathogens (i.e., xenophagy) and is also involved in regulating inflammatory and immune responses. Regulation of lipid metabolism (lipophagy) is a newly identified function of autophagy that may be important in liver and metabolic diseases. Choi AM et al. N Engl J Med 2013;368:

30 Figure 3. Enumeration of intracellular Mtb in lung phagocytes.Repasy T, Lee J, Marino S, Martinez N, et al. (2013) Intracellular Bacillary Burden Reflects a Burst Size for Mycobacterium tuberculosis In Vivo. PLoS Pathog 9(2): e doi: /journal.ppat

31 Median (±SD) Log10 Count of Colony-Forming Units (CFUs)Figure 3. Median (±SD) Log10 Count of Colony-Forming Units (CFUs). Median (±SD) log10 CFU counts over time are shown in the subgroup of 22 patients who provided pooled-sputum samples. Diacon AH et al. N Engl J Med 2009;360:

32 Kaplan–Meier Curves for Culture Conversion According to Time since Randomization.Figure 2 Kaplan–Meier Curves for Culture Conversion According to Time since Randomization. Panel A shows the results for solid culture medium, and Panel B the results for liquid culture medium. In both panels, the gray vertical lines indicate the start of treatment (at 2 months) in the delayed-treatment group and the time of data censoring (at 4 months). Panel C shows the time to culture conversion on solid medium (solid line) along with the 95% confidence interval (dashed lines) for the 38 participants who received linezolid, according to the duration of linezolid therapy. Tick marks indicate the censored observations at the time of the last follow-up visit with culture results. Lee M et al. N Engl J Med 2012;367:

33 Situación actual

34 Probability of Event-free Survival over Time.Figure 3 Probability of Event-free Survival over Time. The Kaplan–Meier curves in Panel A show the time to the onset of clinically significant adverse events that resulted in a drug holiday or dose adjustment during the study. Symbols indicate data-censoring points for patients remaining in the study (see Table 3 in the Supplementary Appendix for detailed risk estimates corrected for person-years of exposure). The curves in Panel B show the time to the first adverse event in patients after the second randomization to either a continuation of the 600-mg daily dose or a reduced dose of 300 mg per day. Tick marks indicate data-censoring points for individual patients who continued to receive the study drug. Lee M et al. N Engl J Med 2012;367:

35 Evaluate at 15 months: Proportion of patients with SCCESTUDIOS FASE III MDR TMC-C210 NCT Brazil, Cambodia, China, Colombia, Estonia, Korea, Latvia, Mexico, Peru, Phillipines, Russia, S Africa, Taiwan, Turkey, Ukraine, Vietnam M (pre-X)DR-TB HIV −/+ 9OB+J 9OB+placebo Evaluate at 15 months: Proportion of patients with SCC Otsuka NCT Estonia, India, Latvia, Lithuania, Moldova, Peru, Phillipines, S Africa MDR-TB HIV− (HIV+ sub-study) 4OB+D 4OB+placebo Evaluate at 2 and 6 months: Proportion of patients with SCC Time to SCC

36 Fármacos inductores de autofagia para controlar la infección por MFármacos inductores de autofagia para controlar la infección por M. tuberculosis Instituto Nacional de Enfermedades Respiratorias “Ismael Cosío Villegas”

37 Pharmacological induction of autophagy in macrophages infected with Mycobacterium tuberculosisJuárez E., Carranza C., Chávez J., Torres M. and Sada E. National Institute of Respiratory Diseases. Mexico City Tuberculosis is a major cause of morbidity and mortality in the world today. M. tuberculosis (Mtb) evade the phagosome-lyosome pathway and escape from macrophages bactericidal responses. Autophagy is a complex process that ultimately generates a degradative vesicle called autophagosome. Autophagy has been demonstrated to participate in the control of Mtb infection. Different drugs can induce autophagy, the best known is the immunosupresor drug Rapamycine but different drugs used for other medical problems have demonstrated to induce autophagy. These drugs include Nitazoxamide, Carbamazepine, Loperamide and Valproic Acid. In this project, we used an in vitro model of autophagy in human monocyte derived macrophages (MDM) and primary murine alveolar macrophages (AM) that were infected with Mtb H37Rv; we further examined the induction of autophagy and the containment of Mtb within the autophagosome. To evaluate the induction of autophagy by Nitazoxanide, Carbamazepine, Loperamide and Valproic Acid in human MDM and murine AM with and without Mtb infection. Autophagosomes detection by flow cytometry and fluorescence microscopy (LC3 and/or ATG16L1). Colocalization of Mtb with LC3 and ubiquitin. Gene expression of LC3 and IRGM by qPCR. MDM/AM 24 h Autophagy evaluation Drug Mtb 5:1 Treatment with Rapamycine 250 ng/ml, Loperamide 3 mM, Valproic Acid 1 mM, and Carbamazepine 0.5 mM induced autophagy in MDM. Nitazoxanide did not induce autophagy at concentrations between 1 to 50 mM. qPCR analysis confirmed that all four drugs increased gene expression of typical autophagy marker LC3 (Fig. 1). Autophagy was confirmed by inmunofluorescence microscopy (presence of LC3+ puncta in Fig. 2). In infected MDM, Mtb colocalized with LC3 and ubiquitin following treatment with rapamycin, loperamide, carbamazepine and valproic acid (Fig.3). Rapamycin, loperamide and carbamazepine induced autophagy in alveolar macrophages from mouse lung homogenates. Infected AM also showed confinement of Mtb within autophagosomes after treatment (Fig.4). Loperamide, valproic acid and carbamazepine were inducers of autophagy that helped contain Mtb within autophagosomes in human and murine macrophages. These results are a first step of a project that evaluates pharmacological induction of autophagy as adjuvant therapy for tuberculosis. A MEDIUM RAPAMYCINE LOPERAMIDE ATG16L1 HOESCHT LC3 B Mtb Mtb + Loperamide Mtb + Carbamazepine HOESCHT, LC3 MTB, MTB-LC3+ Phase contrast C CARBAZEPINE Figure 2. Autophagy induction in MDM. Cells were stimulated with rapamycine (250 ng/ml), carbamazepine (0.5mM) and loperamide (3mM) and valproic acid (1mM, not depicted) for 24h. A) The presence of LC3+ vesicles (puncta) evidenced autophagy. Cells were stained with anti-LC3-FITC and nucleus with Hoescht. Magnification 1000x. B) Phase contrast image, 1000x. C) Percentage of cells with LC3 puncta, medians are indicated. Figure 3. Autophagy induction in infecetd MDM. Cells were infected with Mtb H37Rv for 24h followed by treatment with rapamycine (250 ng/m), carbamazepine (0.5mM) and loperamide (3mM), nitaxozanide (10mM) and valproic acid (1mM) for another 24h. A) The presence of LC3+ vesicles was evaluated and quantitated. Cells were stained with anti-LC3-FITC, Mtb and ubiquitin (not depicted) were detected with specific antibodies revealed with Alexa 495 and nucleus with Hoescht. Magnification 1000x. B) Colocalization of Mtb with LC3 (B) and ubiquitin (C), medians are indicated. Figure 4. Induction of autophagy in murine alveolar macrophages (AM). AM were isolated from lung homogenates (A) and stimulated with rapamycine, carbamazepine and loperamide for 24h, autophagy was detected using anti ATGC16L1-DayLight 488 or LC3-FITC .Magnification 100x. (B). AM were infected with MtbH37Rv for 24h followed by treatment with the indicated drugs for another 24h. Confinement of Mtb within autophagosomes was detected as described in fig.3 legend (C). Representative of 4 independent experiments. Figure 1. Capability of the study drugs to induce autophagy in human macrophages. Cells were stimulated with increased concentrations of the indicated drugs for 24h and the formation of vesicles was determined by flow cytometry. A) Median fluorescence intensity relative to the unstimulated cells. B) LC3 gene expression relative to that of unstimulated cells measured by qPCR. Mean ± SE of 3 independent experiments. Carbamazepine Loperamide Valproic acid Fold change LC3 gene expression mM Nitaxozanide Rapamycine Median fluorescence intensity relative to that of unstimulted cells ng/ml Introduction Objective Results Methods Conclusion B Mtb + Loperamide Mtb + Carbamazepine Mtb Mtb + Rapamycine HOESCHT, LC3 MTB, MTB-LC3+ C

38 Figura 1.Los fármacos inducen un aumento en el número de macrófagos humanos que realizan autofagiaLOPERAMIDE LC3-DAYLIGHT 488 RAPAMYCINE MEDIUM HOESCHT VERAPAMIL VALPROIC ACID CARBAMAZEPINE SIMVASTATIN MEDIUM, phase contrast B C

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40 ESTUDIOS FASE III ACORTADOS 4 MESESPROYECTO PAIS POBLACION ESQUEMA OBJETIVOS RIFAQUIN S Africa, Zimbabwe, Zambia, Mozambique DS-TB HIV−/+(CD4>200/μl) 2RMZE→2Rp15/20mg/kgM[x2/wk] 2RHZE→4RH Evaluate at 18 months: Clinical failure/relapse OFLOTUB NCT Benin, Guinea, Kenya, Senegal, S Africa DS-TB HIV−/+ 2RHPG→2RHG 2RHZE→4RH Evaluate at 30 months: Clinical failure/relapse REMoxTB NCT China, India, Kenya, Malaysia, Mexico, S Africa, Tanzania, Thailand, Zambia 2RHZM→2RHM 2RMZE→2 RM 2RHZE→4RH

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42 Original Article Linezolid for Treatment of Chronic Extensively Drug-Resistant TuberculosisMyungsun Lee, M.D., Jongseok Lee, Ph.D., Matthew W. Carroll, M.D., Hongjo Choi, M.D., Seonyeong Min, R.N., Taeksun Song, Ph.D., Laura E. Via, Ph.D., Lisa C. Goldfeder, C.C.R.P., Eunhwa Kang, M.Sc., Boyoung Jin, R.N., Hyeeun Park, R.N., Hyunkyung Kwak, B.S., Hyunchul Kim, Ph.D., Han-Seung Jeon, M.S., Ina Jeong, M.D., Joon Sung Joh, M.D., Ray Y. Chen, M.D., Kenneth N. Olivier, M.D., Pamela A. Shaw, Ph.D., Dean Follmann, Ph.D., Sun Dae Song, M.D., Ph.D., Jong-Koo Lee, M.D., Dukhyoung Lee, M.D., Cheon Tae Kim, M.D., Veronique Dartois, Ph.D., Seung-Kyu Park, M.D., Sang-Nae Cho, D.V.M., Ph.D., and Clifton E. Barry, III, Ph.D. N Engl J Med Volume 367(16): October 18, 2012

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44 Bedaquiline is the first new TB drug since the introduction of rifampin in 1970.