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THIORIDAZINE CURES MDR/XDR TB INFECTIONS. Targetting the human macrophage for enhanced killing of intracellular mdr/xdr mtb: a new concept for therapy.

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Presentation on theme: "THIORIDAZINE CURES MDR/XDR TB INFECTIONS. Targetting the human macrophage for enhanced killing of intracellular mdr/xdr mtb: a new concept for therapy."— Presentation transcript:

1 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) ISN

2 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 Mycobacterium tuberculosis

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

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

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

7 L Amaral, June 2012 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!

8 L Amaral, June 2012 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). DEFINITION OF ANTIBIOTIC RESISTANCE

9 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.

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

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

12 L Amaral, June 2012 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 ?????

13 L Amaral, June 2012 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 ?????

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

15 L Amaral, June 2012 ESSENTIAL CHARACTERISTICS FOR A PERFECT ANTI-MDR/XDR/TDR-TB DRUG! 1.HAVE ACTIVITY AGAINST ALL FORMS OF ANTIBIOTIC RESISTANT STRAINS. 2.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.

16 L Amaral, June 2012 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).

17 189119531964 TZ DERIVATIVES 2004-2008 Guttman & Erhlich Charpentier Schnetzler & Carrel Amaral et al; others Serious side-effects (toxicity) Gradually replaced by TZ (TZ) (CPZ) Mellaril ® less toxic than CPZ Drowsiness (most common) Wide gamut of in vitro antimicrobial activity. Develop new anti-TB drugs effective against intracellular MDR/XDR-TB. Phenothiazines

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

19 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, 4 th November 2008 TZ

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

21 L Amaral, June 2012 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!

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

23 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. TZ + TZ derivatives

24 L Amaral, June 2012 In Vitro Growth inhibition of Mycobacterium tuberculosis by Thioridazine derivatives. Compounds MIC 50 (mg/L). Thioridazine 10 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

25 L Amaral, June 2012 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.

26 L Amaral, June 2012 INTRACELLULAR ACTIVTY OF NON-PHENOTHIAZINES

27 L Amaral, June 2012 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

28 L Amaral, June 2012 IN VIVO STUDIES

29 E F 2550100200400 1200 mg TZ/kg/day 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 Animal model: Balb/C females (25 gms) Toxicity assays - TZ

30 100Control400 1200 mg TZ/kg/day Day 0 I.P. injection Day 30 Treatment (TZ) Infection with TB (1×10 6 CFU/mL) Except the Control group Organs removal (lungs, liver and spleen) – plating – CFU NO TZ Infection studies with M. tuberculosis

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

32 CONFIRMATION OF MOUSE WORK van 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.

33 Thioridazine in combination with INH, Rifampin and PZA. L Amaral June 2012 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.

34 H37Rv Control infection H37Rv Infection Plus Thioridazine MDR Control Infection MDR Infection Plus Thioridazine (A) Extensive lung consolidation (arrows) is visible in control animals after 120 days of infection by drug- sensitive control Strain H37Rv. (B) In contrast, less pneumonia (arrow) is seen in the lung of Mice treated with Thioridazine 32 mg/kg daily by intragastric cannula. (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

35 Thioridazine cures XDR TB L Amaral June 2012

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37 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 Antimicrob Chemother. 2011 Dec 1.[Epub ahead of print] PubMed PMID: 22134348.

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39 MECHANISM OF ACTION L Amaral June 2012

40 Inhibition of macrophage intracellular pumps? Concentration of the compound inside the macrophage? 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 Ca 2+ / K + pumps inhibitors Inhibitors of Ca 2+ transport (calmodulin) and influx/efflux processes dependent on cellular energy 0.1 mg/L 10 mg/L Clinical concentrations However… Questions raised… Mechanism of action of TZ and its derivatives inside the macrophage?

41 D ependent upon transport processes affected by agents that inhibit Ca 2+ -activated K + pumps Killing activity of phagocytic cells (Neutrophils) Correlated with K + availability Rise of Ca 2+ and K + associated with the killing activity of phagocytic cells Other studies…

42 Inhibition of efflux pumps of bacteria and eukaryotic cells indirect effects on the K + pumps of mammalian cells direct effects on the K + pumps of mammalian cells Ca 2+ channel blocker; inhibits plasma membrane mediated transport of K + into the macrophage preventing access to Ca 2+ Verapamil Reserpine Ouabain Ca 2+ /K + pump inhibitors Other Ca 2+ /K + pump inhibitors

43 Enhancement of the macrophage killing activity Reduction of CFU with all the inhibitors tested Martins M & Amaral L (2006). Res. J. Microbiol. 1(3): 203. In phagocytosed MRSA: In phagocytosed M. tuberculosis: Enhancement of the macrophage killing activity Reduction of CFU Verapamil: more active than TZ (higher concentrations) MACROPHAGE MODEL MRSA Results – Ex vivo studies with the other Ca 2+ /K + pump inhibitors Martins M, Viveiros M, Amaral L. (2008). In Vivo 22(1): 69. M. tuberculosis

44 K+ H+ Ca 2+ H+ K+ ATP K+ ATP Ca 2+ ATP Na+ K+K+ Ca 2+ ATP H+H+ K+K+ Ca 2+ Na+ B K+ ATP Ca 2+ K+ Ca 2+ H+ ATP H+ E G K+ H+ Ca 2+ K+ Ca 2+ ATP K+ Ca 2+ K+ H+ Ca 2+ ATP K+ H+ ATP C D H+H+ K+K+ H+H+ Ca 2+ H+H+ K+K+ F ATP K+ H+ Ca 2+ Amaral L, Martins M, Viveiros M. (2007). J. Antimicrob. Chemother. 59:1237. H+H+ H+H+ H+H+ ATP K+K+ Ca 2+ K+K+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ A L Amaral, May 2011

45 The Role of efflux pumps in MDR-Mtb

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

47 L Amaral, June 2012 Time course of induced INH resistance and reversion of the H37Rv (ATCC 27294) reference strain. 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. RE Thioridazine added @ IL 7 immediately reduces resistance to INH (arrow)

48 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. transport of fluorescent substrates (EB) through the cell envelope of living bacterial cells common substrate of bacterial efflux pumps emits weak fluorescence in aqueous solution (outside cells) and becomes strongly fluorescent when concentrated in periplasm Automated EB method for bacteria

49 Detection of Efflux Activity by Real-time Fluorometry  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) Ethidium bromide (EtBr) LED light source (rotates for each channel) rotor spins tubes at 500 rpm lens filter set (rotates for each channel) sensitive PMT (photomultiplier) detector Rotor-Gene 3000 TM (Corbett Research) PKI

50 Effect of chlorpromazine (CPZ), thioridazine (TZ) and verapamil (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.

51 Strains Relative expression level ± SD mmpl7Rv1258cp55efpAmmrRv2459 H37Rv INH (R)0.441.990.820.340.440.50 H37Rv INH (I)10.5615.266.968.009.9522.63 401/06 INH (C)34.3022.6318.3816.0024.259.19 401/07 INH (C)17.1514.939.856.969.1927.86 401/08 INH (C)4.167.8011.318.572.292.64 Average quantification of the relative expression level, by RT-qPCR, of the genes that code for efflux pumps in M. tuberculosis. Machado D, Couto I, Perdigão J, Rodrigues L, Portugal I, Baptista P, Veigas B, Viveiros M. and Amaral L. Contribution of efflux to the emergence of isoniazid and multidrug resistance in Mycobacterium tuberculosis. PLoS One 2012; 7(4): e34538. doi:10.1371/journal.pone.0034538

52 CONCLUSIONS THIORIDAZINE HAS IN VITRO ACTIVITY AGAINST ALL STRAINS OF Mtb.THIORIDAZINE HAS IN VITRO ACTIVITY AGAINST ALL STRAINS OF Mtb. THIORIDAZINE ENHANCES INTRACELLULAR KILLING OF MDR/XDR Mtb.THIORIDAZINE ENHANCES INTRACELLULAR KILLING OF MDR/XDR Mtb. THIORIDAZINE CURES THE MOUSE OF AN MDR Mtb INFECTION.THIORIDAZINE CURES THE MOUSE OF AN MDR Mtb INFECTION. THIORIDAZINE CURES THE HUMAN OF AN XDR-TB INFECTION.THIORIDAZINE CURES THE HUMAN OF AN XDR-TB INFECTION. DUAL MECHANISM OF ACTION:DUAL MECHANISM OF ACTION: A) INHIBITION OF K+ EFFLUX OF MACROPHAGE PERMITTING ACIDIFICATION OF PHAGOLYSOSOME. A) INHIBITION OF K+ EFFLUX OF MACROPHAGE PERMITTING ACIDIFICATION OF PHAGOLYSOSOME. B) INHIBITION OF EFFLUX PUMPS OF Mtb THAT BESTOW MDR PHENOTYPE. B) INHIBITION OF EFFLUX PUMPS OF Mtb THAT BESTOW MDR PHENOTYPE. BY-PASSES ANY MUTATIONAL RESPONSE MADE BY Mtb THAT WOULD LEAD TO RESISTANCE.BY-PASSES ANY MUTATIONAL RESPONSE MADE BY Mtb THAT WOULD LEAD TO RESISTANCE. THIORIDAZINE IS CHEAP, RELATIVELY SAFE WHEN PATIENT IS MONITORED FOR CARDIA FUNCTION, AND SHOULD BE CONSIDERED FOR THERAPY OF XDR/TDR Mtb INFECTIONS.THIORIDAZINE IS CHEAP, RELATIVELY SAFE WHEN PATIENT IS MONITORED FOR CARDIA FUNCTION, AND SHOULD BE CONSIDERED FOR THERAPY OF XDR/TDR Mtb INFECTIONS.

53 May 2011 Special Issue of PRI: Thioridazine MDR/XDR TB

54 ADDITIONAL BENEFITS OF THIORIDAZINE  INHIBITS ESSENTIAL GENES OF Mtb (Dutta NK, Mazumdar K, Dastidar SG, Karakousis PC, Amaral L. New patentable use of an old neuroleptic compound thioridazine to combat tuberculosis: a gene regulation perspective. Recent Pat Antiinfect Drug Discov 2011;6:128-138.)  IMPROVES QUALITY OF LIFE OF THE XDR-TB PATIENT (Udwadia ZF, Sen T, Pinto LM. Safety and efficacy of thioridazine as salvagetherapy in Indian patients with XDR-TB. Recent Pat Antiinfect Drug Discov. 2011;6:88-91.  KILLS DORMANT Mtb (Sohaskey C. Latent tuberculosis: is there a role for thioridazine? Recent Pat Antiinfect Drug Discov. 2011;6:139-46. Review. ).  ANTICIPATED SHORT TERM USAGE FOR MDR/XDR/TDR-TB INFECTIONS WILL DO NO HARM (Thanacoody RH. Thioridazine: the good and the bad. Recent Pat Antiinfect Drug Discov 2011;6:92-98. Review.).

55 RESEARCH TEAM L Amaral, May 2011 ISN


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