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THIORIDAZINE CURES MDR/XDR TB INFECTIONS .

ISN. THIORIDAZINE CURES MDR/XDR TB INFECTIONS . Targetting the human macrophage for enhanced killing of intracellular mdr/xdr mtb: a new concept for therapy of MDR/XDR TB. Leonard Amaral (Portugal) Eduardo Abbate (Argentina) Martin J Boeree (Netherlands)

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THIORIDAZINE CURES MDR/XDR TB INFECTIONS .

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  1. ISN THIORIDAZINE CURES MDR/XDR TB INFECTIONS. Targetting the human macrophage for enhanced killing of intracellular mdr/xdr mtb: a new concept for therapy of MDR/XDR TB. Leonard Amaral (Portugal) Eduardo Abbate (Argentina) Martin J Boeree (Netherlands) Noton Dutta (USA) Paulo Ferrinho (Portugal) Stephen H Gillespie (UK) Eduardo Gotuzzo (Peru) Jette E Kristiansen (Denmark) Marta S Martins (Ireland) Chuck Sohaskey (USA) Rubin Thanacoody (UK) Zarir F Udwadia (India) Jakko van Ingen (Netherlands) Dick van Soolingen (Netherlands) Miguel Viveiros (Portugal)

  2. Mycobacterium tuberculosis • Causative agent of Tuberculosis (TB) • Emergence of Multi-Drug Resistant strains (MDR-TB) Resistance to at least isoniazid (INH) + rifampicin (RIF) • Emergence of Extensively-Drug Resistance strains (XDR-TB) WHO; Weekly Epidemiol Record; 2008

  3. Progression of Resistance TOTALLY DRUG RESISTANT Mtb Remains to be defined

  4. New approaches - Phenothiazines Purpose of the studies In vitro, ex vivo and in vivo studies Macrophage model Multi-Drug Resistant M. tuberculosis Role of efflux pumps

  5. PULMONARY TUBERCULOSIS IS CAUSED BY THE STEADFAST HUMAN PATHOGEN MYCOBATERIUM tuberculosis. IT IS AN INTRACELLULAR INFECTION OF THE PULMONARY MACROPHAGE (ALVEOLAR II CELL) L Amaral, June 2012

  6. WHEREAS AN INFECTION BY Mtb IS INTRACELLULAR, ACTIVE TUBERCULOSIS IS WHEN THE BACTERIUM BREAKS OUT OF ITS MACROPHAGE PRISON AND IS EXPELLED TO THE OUTSIDE IN MICRODROPLETS OF SPUTUM. ACTIVE TB IS INFECTIOUS! L Amaral, June 2012

  7. DEFINITION OF ANTIBIOTIC RESISTANCE ANTIBIOTIC SUSCEPTIBLE Mtb SUSCEPTIBLE TO ISONIAZID (INH) AND RIFAMPIN (RF). MULTIDRUG RESISTANT Mtb (MDR-TB) RESISTANT TO INH AND RF. EXTENSIVELY DRUG RESISTANT Mtb (XDR TB) RESISTANT TO INH, RF, STREPTOMYCIN, ANY FLUOROQUINOLONE, AND TO INJECTAB LE ANTI-TB DRUGS (AMIKACIN, KANAMYCIN AND CAPREOMYCIN). L Amaral, June 2012

  8. TDR-TB 5 CASES OF TDR-TB IN ONE MUMBAI HOSPITAL 2012. IS THERE A PROBLEM? Udwadia ZF. Totally drug resistant tuberculosis in India: who let the djinn out? Respirology. 2012. [Epub ahead of print] PubMed PMID: 22564108.

  9. W.H.O 2009 SHORT GLOBAL REPORT L Amaral, June 2012

  10. PROBLEM: WHAT IS MEANT BY NEW CASES OF TUBERCULOSIS? ANSWER: NEW CASES OF ACTIVE TB L Amaral, June 2012

  11. GLOBAL TUBERCULOSIS: FACTS 2007 (W.H.O. 2009 Report). 2 TO 3 BILLION INFECTED WITH Mtb. 10.5 MILLION NEW CASES. OVER 2 MILLION DEATHS. 511,000 NEW CASES OF MDR TB. XDR TB ????? TDR-TB ????? L Amaral, June 2012

  12. MORTALITY PRODUCED BY: MDR-TB 20 TO 80 % within 2 years. MDR-TB co-infected with HIV/presenting w/AIDS 80 to 100 % within 1 year depending on area where therapy is given. XDR-TB > 80% within 6 months or sooner if co-infected with HIV/presenting with AIDS. TDR-TB ????? L Amaral, June 2012

  13. THERE ARE NO SAFE AND EFFECTIVE DRUGS AGAINST MDR/XDR/TDR-TB, EXCEPT FOR ----------. L Amaral, June 2012

  14. ESSENTIAL CHARACTERISTICS FOR A PERFECT ANTI-MDR/XDR/TDR-TB DRUG! • HAVE ACTIVITY AGAINST ALL FORMS OF ANTIBIOTIC RESISTANT STRAINS. • HAVE ACTIVITY WHERE THE MDR/XDR TB STRAIN RESIDES: THE HUMAN PULMONARY MACROPHAGE! • 3. HAVE NO TOXICITY. • 4. NO RESISTANCE TO THE DRUG CAN BE MADE BY MUTATION OF MDR/XDR/TDR Mtb. L Amaral, June 2012

  15. HISTORY OF PHENOTHIAZINES 1890 MB inhibits mobiliby of bacteria; kills malaria and syphilis causing organisms (Guttmann & Ehrlich). 1890’s Makes cats and humans lethargic (Bodini). 1957 Methylene blue (MB) dye. Chlorpromazine (CPZ) derived from MB by Rhone-Polenc: first neuroleptic. 1975 CPZ In Vitro activity against Mtb (Molnar). 1966 Thioridazine (TZ) derived from CPZ. 1996 In vitro activity of TZ against all forms of antibiotic resistant strains of Mtb (Amaral et al). L Amaral, June 2012

  16. Develop new anti-TB drugs effective against intracellular MDR/XDR-TB. Phenothiazines TZ DERIVATIVES Amaral et al; others • Mellaril® • less toxic than CPZ • Drowsiness (most common) Schnetzler & Carrel (TZ) Charpentier • Serious side-effects (toxicity) • Gradually replaced by TZ (CPZ) Guttman & Erhlich • Wide gamut of in vitro antimicrobial activity. 1891 1953 1964 2004-2008

  17. Thioridazine (TZ) Chlorpromazine (CPZ) PHENOTHIAZINES L Amaral, June 2012

  18. TZ The antimycobacterial activity of thioridazine derivatives against drug resistant Mycobacterium tuberculosis: in vitro, ex vivo and in vivo studies Unidade de Micobactérias and UPMM Instituto de Higiene e Medicina Tropical Universidade Nova de Lisboa Lisbon, 4th November 2008

  19. Amaral L et al..Journal of Antimicrobial Chemotherapy (1996) 38, 1049-1053 L Amaral, June 2012

  20. HOWEVER, IN VITRO CONCENTRATIONS ARE CLINICALLY IRRELEVANT SINCE THE MAXIMUM CONCENTRATION OF THESE COMPOUNDS THAT CAN BE ACHIEVED IN THE PATIENT IS 0.5 mg/L OF PLASMA! L Amaral, June 2012

  21. Experimental outline Screening of TZ derivatives (22) Mutagenicity Assay (Ames test) Toxicity in lymphocytes (Trypan blue exclusion method) Non-toxic and non-mutagenic derivatives Studies S. aureus / MRSA (model) MIC and MBC – microdilution method In vitro M. tuberculosis / MDR-TB MIC and MBC – BACTEC 460TM Ex vivo Minimum Inhibitory Concentration (MIC) Minimum Bactericidal Concentration (MBC) Infection studies in macrophages (phagocytosis assay) In vivo Toxicity assays (Balb/C) – TZ and derivatives Infection studies – TZ and derivatives

  22. TZ derivatives TZ + • Synthesis: Prof. György Hajós (Chemistry Institute of Budapest, Hungary); Chemical manipulation of the parent compound (TZ) Amaral L, Martins M and Viveiros M. (2007). Infect. Disord. Drug Targets 7(3):257.

  23. In Vitro Growth inhibition of Mycobacterium tuberculosis by Thioridazine derivatives. Compounds MIC 50 (mg/L). Thioridazine10 Isoniazid 0.08 #1550 >20 #1686 20 #1687 20 #1532 20 #1688 20 #1689 20 #1819 >20 #1820 >20 #1821 >20 #1867 5 #1868 20 #1869 >20 #1870 10 #1871 >20 #1872 20 #1873 >20 #1874 >20 #1875 5 #1929 >20 L Amaral, June 2012

  24. ENHANCED KILLING OF MYCOBACTERIUM tuberculosis by 0.1 mg/L of DERIVATIVES OF THIORIDAZINE. Martins M, Schelz Z, Martins A, Molnar J, Hajös G, Riedl Z, Viveiros M, Yalcin I, Aki-Sener E, Amaral L.. In vitro and ex vivo activity of thioridazine derivatives against Mycobacterium tuberculosis. Int J Antimicrob Agents. 2007;;29:338-40. L Amaral, June 2012

  25. INTRACELLULAR ACTIVTY OF NON-PHENOTHIAZINES L Amaral, June 2012

  26. ENHANCED KILLING ACTIVITY OF XDR-TB BY SILA 421 Martins M, Viveiros M, Ramos J, Couto I, Molnar J, Boeree M, Amaral L. SILA 421, an inhibitor of efflux pumps of cancer cells, enhances the killing of intracellular extensively drug-resistant tuberculosis (XDR-TB). Int J Antimicrob Agents. 2009;33:479-82 L Amaral, June 2012

  27. IN VIVO STUDIES L Amaral, June 2012

  28. Toxicity assays - TZ F E • Animal model: Balb/C females (25 gms) mg TZ/kg/day 1200 25 50 100 200 400 • daily inoculations • daily weighing √ In all the concentrations tested no toxicity was obtained √ Concentrations selected for treatment: 100, 400 and 1200 mg TZ/Kg/day

  29. Infection studies with M. tuberculosis Infection with TB (1×106 CFU/mL) Day 0 I.P. injection mg TZ/kg/day Day 30 Treatment (TZ) NO TZ Except the Control group 1200 Control 100 400 • Organs removal (lungs, liver and spleen) – plating – CFU

  30. Results - In vivo studies Lungs Liver Spleen Mice treated with TZ (equivalent in the human to 1200 mg/Kg/day) • colony forming units (CFU) reduction – lungs Amaral L, Martins M, Viveiros M. (2007). J. Antimicrob. Chemother. 59:1237.

  31. CONFIRMATION OF MOUSE WORKvan Soolingen D, Pando RH, Orozco H, Aguilar D, Magis C, van Ingen J, Amaral L, Boeree M. Thioridazine shows promising activity in a murine model of multi-drug resistant tuberculosis. PloS One 2010;5. pii: e12640.

  32. Thioridazine in combination with INH, Rifampin and PZA. Effect of combined treatment with standard anti-tuberculosis treatment and TZ on lung bacillary load in mice infected with M. Tuberculosis H37Rv Grey bars: Animals treated 60 days with conventional therapy: Isoniazid, Rifampicin and Pyrazinamide Black bars: Animals treated 60 days with conventional therapy in combination with TZ 32mg daily White bars: untreated control mice. L Amaral June 2012

  33. H37Rv Infection Plus Thioridazine H37Rv Control infection (B) In contrast,less pneumonia (arrow) is seen in the lung of Mice treated with Thioridazine 32 mg/kg daily byintragastric cannula. (A) Extensive lung consolidation (arrows) is visible in control animals after 120 days of infection by drug-sensitive control Strain H37Rv. MDR Infection Plus Thioridazine MDR Control Infection (C) Control mice after 120 days of infection with MDR strain show extensive pneumonic areas (arrow) In comparison, less lung consolidation (arrow) is seen in the lung of mice infected by the MDR-TB strain and treated daily during two months with 70 mg/kg of thioridazine L Amaral June 2012

  34. Thioridazine cures XDR TB L Amaral June 2012

  35. L Amaral June 2012

  36. CURE OF XDR-TB (SECOND STUDY) • Abbate E, Vescovo M, Natiello M, Cufré M, García A, Gonzalez Montaner P,Ambroggi M, Ritacco V, van Soolingen D. Successful alternative treatment of extensively drug-resistant tuberculosis in Argentina with a combination of linezolid, moxifloxacin and thioridazine. J AntimicrobChemother. 2011 Dec 1.[Epub ahead of print] PubMed PMID: 22134348.

  37. MECHANISM OF ACTION L Amaral June 2012

  38. Mechanism of action of TZ and its derivatives inside the macrophage? Concentration of the compound inside the macrophage? Inhibition of macrophage intracellular pumps? Questions raised… • macrophage can concentrate 100 times the phenothiazines inside lysosomes / phagosomes • MIC TZ (TB/MDR-TB) = 20 mg/L • MBC TZ (TB/MDR-TB) = 30 mg/L • Ca2+ / K+ pumps inhibitors • Inhibitors of Ca2+ transport (calmodulin) and influx/efflux processes dependent on cellular energy • Clinical concentrations 0.1 mg/L 10 mg/L However…

  39. Other studies… • Killing activity of phagocytic cells (Neutrophils) • Correlated with K+ availability • Dependent upon transport processes affected by agents that inhibit Ca2+-activated K+ pumps • Rise of Ca2+ and K+ associated with the killing activity of phagocytic cells

  40. Other Ca2+/K+ pump inhibitors Ca2+/K+ pump inhibitors Verapamil Ouabain Reserpine • Inhibition of efflux pumps of bacteria and eukaryotic cells • indirect effects on the K+ pumps of mammalian cells • Ca2+ channel blocker; inhibits plasma membrane mediated transport of K+ into the macrophage preventing access to Ca2+ • direct effects on the K+ pumps of mammalian cells

  41. Results – Ex vivo studies with the other Ca2+/K+ pump inhibitors MRSA In phagocytosed MRSA: • Enhancement of the macrophage killing activity • Reduction of CFU with all the inhibitors tested M. tuberculosis Martins M & Amaral L (2006). Res. J. Microbiol. 1(3): 203. In phagocytosed M. tuberculosis: • Enhancement of the macrophage killing activity • Reduction of CFU • Verapamil: more active than TZ (higher concentrations) • MACROPHAGE MODEL Martins M, Viveiros M, Amaral L. (2008). In Vivo 22(1): 69.

  42. A K+ H+ B H+ ATP Ca2+ Ca2+ Ca2+ Ca2+ K+ C H+ K+ H+ ATP ATP Ca2+ H+ Ca2+ K+ Ca2+ K+ Ca2+ Ca2+ K+ Ca2+ Ca2+ H+ H+ K+ Ca2+ ATP Ca2+ ATP K+ H+ Ca2+ K+ H+ Ca2+ H+ H+ H+ K+ H+ K+ H+ H+ H+ H+ Ca2+ E ATP Ca2+ H+ Ca2+ ATP Ca2+ K+ Ca2+ H+ Ca2+ D Ca2+ ATP G Ca2+ Ca2+ F K+ Ca2+ Na+ ATP H+ K+ Ca2+ H+ K+ Ca2+ ATP Ca2+ Ca2+ Ca2+ Ca2+ ATP ATP K+ H+ ATP Ca2+ K+ Ca2+ Ca2+ Na+ K+ Ca2+ H+ ATP L Amaral, May 2011 Amaral L, Martins M, Viveiros M. (2007). J. Antimicrob. Chemother. 59:1237.

  43. The Role of efflux pumps in MDR-Mtb

  44. Laboratory demonstrations of induced efflux activity by bacteria. L Amaral, June 2012

  45. Time course of induced INH resistance and reversion of the H37Rv (ATCC 27294) reference strain. Thioridazine added @ IL 7 immediately reduces resistance to INH (arrow) RE Viveiros M, Portugal I, Bettencourt R, Victor TC, Jordaan AM, Leandro C, Ordway D, Amaral L. Isoniazid-induced transient high-level resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2002 Sep;46(9):2804-10. L Amaral, June 2012

  46. Automated EB method for bacteria Ethidium Bromide (EB) Viveiros M et al, Int J Antimicrob Agents. 31(5):458-62, 2008. Spengler G et al, Anticancer Research 29: 2173-2177, 2009.

  47. rotor spins tubes at 500 rpm filter set (rotates for each channel) sensitive PMT (photomultiplier) detector lens Ethidium bromide (EtBr) LED light source (rotates for each channel) Detection of Efflux Activity by Real-time Fluorometry PKI • Detection of efflux activity on a real-time basis • Separate detection of accumulation and efflux of ethidium bromide (EtBr) • Identification of compounds with efflux pump inhibitory activity – efflux pump inhibitors (EPIs) Rotor-Gene 3000TM (Corbett Research)

  48. Effect of chlorpromazine (CPZ), thioridazine (TZ) andverapamil (VP) on the efflux of ethidium bromide in M. smegmatismc2155 (A) and M. avium ATCC25291T (B) Rodrigues L, Aínsa JA, Amaral L and Viveiros M. Inhibition of Drug Efflux in Mycobacteria with Phenothiazines and Other Putative Efflux Inhibitors. Recent Patents on Anti-Infective Drug Discovery, 2011; 6:118-127.

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