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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)
Noton Dutta (USA)
Stephen H Gillespie (UK)
Jette E Kristiansen (Denmark)
Chuck Sohaskey (USA)
Zarir F Udwadia (India)
Dick van Soolingen (Netherlands)
Martin J Boeree (Netherlands)
Paulo Ferrinho (Portugal)
Eduardo Gotuzzo (Peru)
Marta S Martins (Ireland)
Rubin Thanacoody (UK)
Jakko van Ingen (Netherlands)
Miguel Viveiros (Portugal)
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
Progression of Resistance
TOTALLY DRUG RESISTANT Mtb
Remains to be defined
Multi-Drug Resistant M. tuberculosis
New approaches - Phenothiazines
Purpose of the studies
In vitro, ex vivo and in vivo studies
Macrophage model
Role of efflux pumps
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
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
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
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.
W.H.O 2009 SHORT GLOBAL REPORT
L Amaral, June 2012
PROBLEM: WHAT IS MEANT BY
NEW CASES OF TUBERCULOSIS?
ANSWER: NEW CASES OF
ACTIVE TB
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 ?????
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 ?????
L Amaral, June 2012
THERE ARE NO SAFE
AND EFFECTIVE
DRUGS AGAINST
MDR/XDR/TDR-TB,
EXCEPT FOR ----------.
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.
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).
L Amaral, June 2012
Develop new anti-TB drugs effective against intracellular MDR/XDR-TB.
Phenothiazines
Amaral et al;
others
TZ
• Mellaril®
DERIVATIVES
• less toxic than CPZ
• Drowsiness (most common)
Schnetzler
& Carrel
(TZ)
Charpentier
• Serious side-effects (toxicity)
(CPZ)
• Gradually replaced by TZ
Guttman
& Erhlich
• Wide gamut of in vitro antimicrobial activity.
1891
1953
1964
2004-2008
PHENOTHIAZINES
Thioridazine (TZ)
Chlorpromazine (CPZ)
L Amaral, June 2012
The antimycobacterial activity of thioridazine derivatives
against drug resistant Mycobacterium tuberculosis:
in vitro, ex vivo and in vivo studies
TZ
Unidade de Micobactérias and UPMM
Instituto de Higiene e Medicina Tropical
Universidade Nova de Lisboa
Lisbon, 4th November 2008
Amaral L et al..Journal of Antimicrobial Chemotherapy (1996) 38, 1049-1053
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!
L Amaral, June 2012
Experimental outline
Screening of TZ derivatives (22)
Mutagenicity Assay
(Ames test)
Toxicity in lymphocytes
(Trypan blue exclusion method)
Non-toxic and non-mutagenic
Studies
derivatives
In vitro
S. aureus / MRSA (model)
MIC and MBC – microdilution method
M. tuberculosis / MDR-TB
MIC and MBC – BACTEC 460TM
Ex vivo
Minimum Inhibitory Concentration (MIC)
Infection studies in macrophages
(phagocytosis assay)
In vivo
Toxicity assays (Balb/C) – TZ and derivatives
Infection studies – TZ and derivatives
Minimum Bactericidal Concentration (MBC)
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.
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
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.
L Amaral, June 2012
INTRACELLULAR ACTIVTY OF
NON-PHENOTHIAZINES
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
L Amaral, June 2012
IN VIVO STUDIES
L Amaral, June 2012
Toxicity assays - TZ
• Animal model: Balb/C females (25 gms)
mg TZ/kg/day
E
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
F
1200
Infection studies with M. tuberculosis
Infection with TB
(1×106 CFU/mL)
Day 0
I.P. injection
mg TZ/kg/day
Day 30
NO TZ
Except the Control group
Treatment (TZ)
Control
100
400
• Organs removal (lungs, liver and spleen) – plating – CFU
1200
Results - In vivo studies
Lungs
Liver
Control
1,00E+10
1200mg TZ
1,00E+08
CFU/mg
1,00E+06
1,00E+04
1,00E+02
1,00E+00
1,00E-02
0
30
60
90
120
150
180
210
240
270
300
Time (days)
Spleen
Control
1,00E+10
1,00E+08
(equivalent in the human to
1,00E+06
1200 mg/Kg/day)
CFU/mg
Mice treated with TZ
1200mg TZ
1,00E+04
1,00E+02
• colony forming units (CFU) reduction – lungs
1,00E+00
1,00E-02
0
30
60
90
120
150
180
Tim e (days)
Amaral L, Martins M, Viveiros M. (2007). J. Antimicrob. Chemother. 59:1237.
210
240
270
300
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.
Thioridazine in combination with INH, Rifampin and PZA.
Effect of combined treatment with standard antituberculosis 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
H37Rv
Control
infection
(A) Extensive lung
consolidation
(arrows) is visible in
control animals after
120 days of
infection by drugsensitive control
Strain H37Rv.
MDR
Control
Infection
(C) Control
mice after 120
days of infection
with MDR strain
show extensive
pneumonic
areas (arrow)
H37Rv
Infection
Plus
Thioridazine
(B) In contrast, less
pneumonia (arrow)
is seen in the lung of
Mice treated with
Thioridazine 32 mg/kg
daily by intragastric cannula.
MDR
Infection
Plus
Thioridazine
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
Thioridazine cures XDR TB
L Amaral June 2012
L Amaral June 2012
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 drugresistant tuberculosis in Argentina with a
combination of linezolid, moxifloxacin and
thioridazine. J Antimicrob Chemother. 2011
Dec 1.[Epub ahead of print] PubMed PMID:
22134348.
MECHANISM OF ACTION
L Amaral June 2012
Mechanism of action of TZ and its derivatives inside the macrophage?
Questions raised…
Concentration of the
compound inside the
macrophage?
• macrophage can concentrate 100 times the
phenothiazines inside lysosomes / phagosomes
• Ca2+ / K+ pumps inhibitors
• Inhibitors of Ca2+ transport (calmodulin) and
influx/efflux
dependent
on
energy
• Clinical concentrations
0.1 mg/L
processes
10 mg/L
However…
Inhibition of
macrophage
• MIC TZ (TB/MDR-TB) = 20 mg/L
• MBC TZ (TB/MDR-TB) = 30 mg/L
intracellular pumps?
cellular
Other studies…
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Article
Nature 416, 291-297 (21 March 2002) | doi:10.1038/416291a; Received 12 December 2001; Acce
January 2002
• Killing activity of phagocytic cells (Neutrophils)
Killing activity of neutrophils is mediated through
activation of proteases by K + flux
Emer P. Reeves1,2, Hui Lu1,2, Hugues Lortat Jacobs3, Carlo G. M. Messina1,
Bolsover4, Giorgio Gabella5, Eric O. Potma6, Alice Warley7, Jürgen Roes8 an
Anthony W. Segal1
1.
2.
3.
4.
5.
6.
• Correlated with
K+
7.
8.
availability
Centre for Molecular Medicine, University College London, 5 University Street, London WC
Centre for Molecular Medicine, Department of Physiology, University College London, 5 Un
Street, London WC1E 6JJ, UK
Centre for Molecular Medicine, Department of Anatomy , University College London, 5 Univ
Street, London WC1E 6JJ, UK
Windeyer Institute of Medical Sciences, University College London, 5 University Street, Lon
6JJ, UK
Institut de Biologie Structurale, 41 rue Horowitz, 38027 Grenoble, France
Ultrafast Laser and Spectroscopy Laboratory, Materials Science Centre, University of Gron
Nijenborgh 4, 9747 AG Groningen, The Netherlands
Rayne Institute, St Thomas' Hospital, Lambeth Palace Road, London SE1 7EH, UK
These authors contributed equally to this work.
Correspondence to: Anthony W. Segal 1 Correspondence and requests for materials should be addre
A.W.S. (e-mail: Email: [email protected]).
Top of page
Abstract
• 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
Other Ca2+/K+ pump inhibitors
Ca2+/K+ pump inhibitors
Verapamil
•
•
Ca2+ channel blocker; inhibits plasma
Ouabain
Reserpine
indirect effects on the K+ pumps of
mammalian cells
membrane mediated transport of K+ into
the macrophage preventing access to Ca2+
• Inhibition of efflux pumps of bacteria and eukaryotic cells
•
direct effects on the K+ pumps
of mammalian cells
Results – Ex vivo studies with the other Ca2+/K+ pump inhibitors
MRSA
Control
Bacterial concentration (bacteria/mL)
1,00E+10
In phagocytosed MRSA:
TZ 0.1 mg/L
1,00E+09
Reserpine 80 mg/L
1,00E+08
Ouabain 80 mg/L
1,00E+07
Verapamil 80mg/L
• Enhancement of the macrophage killing
1,00E+06
activity
1,00E+05
1,00E+04
• Reduction of CFU with all the inhibitors
1,00E+03
tested
1,00E+02
1,00E+01
1,00E+00
0
2
4
6
M. tuberculosis
Hours post-infection
Control
1,00E+07
TZ 0.1 mg/L
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
Bacterial concentration (bacteria/mL)
Reserpine 80 mg/L
1,00E+06
Ouabain 80 mg/L
Verapamil 80 mg/L
1,00E+05
1,00E+04
1,00E+03
1,00E+02
1,00E+01
1,00E+00
0
1
2
3
Days post-infection
Martins M, Viveiros M, Amaral L. (2008). In Vivo 22(1): 69.
K+
Ca2+
A
Na+
ATP
B
Ca2+
H+
K+
Ca2+
Ca2+
K+
Ca2+
H+
K+
Ca2+
H+
C
H+ ATP
K+
ATP
ATP
Ca2+
Ca2+
K+
Ca2+
Ca2+
Ca2+
Ca2+
K+
Ca2+
Ca2+
ATP
K+
Ca2+
H+
ATP
Ca2+
K+
H+
K+
ATP
K+
H+
H+
H+
H+
H+
K+
H+
Ca2+
H+
H+
H+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
G
H+
Ca2+
Ca2+
Ca2+
Amaral L, Martins M, Viveiros M. (2007). J. Antimicrob. Chemother. 59:1237.
F
K+
ATP
D
E
K+
H+
ATP
K+
ATP
Na+
Ca2+
K+
ATP
Ca2+
H+
ATP
Ca2+
Ca2+
H+
ATP
K+
K+
Ca2+
H+
ATP
Ca2+
H+
H+
Ca2+
Ca2+
H+
L Amaral, May 2011
The Role of efflux pumps in
MDR-Mtb
Laboratory demonstrations
of induced efflux activity
by bacteria.
L Amaral, June 2012
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
Automated EB method for bacteria
Ethidium Bromide (EB)
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
Viveiros M et al, Int J Antimicrob Agents. 31(5):458-62, 2008.
Spengler G et al, Anticancer Research 29: 2173-2177, 2009.
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)
Ethidium
bromide (EtBr)
Rotor-Gene 3000TM
(Corbett Research)
rotor spins tubes
at 500 rpm
filter set
(rotates for
each channel)
lens
LED light source
(rotates for each
channel)
sensitive PMT
(photomultiplier)
detector
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.
Average quantification of the relative expression level, by RT-qPCR, of the genes
that code for efflux pumps in M. tuberculosis.
Relative expression level ± SD
Strains
mmpl7 Rv1258c
p55
efpA
mmr
Rv2459
H37Rv INH (R)
0.44
1.99
0.82
0.34
0.44
0.50
H37Rv INH (I)
10.56
15.26
6.96
8.00
9.95
22.63
401/06 INH (C)
34.30
22.63
18.38
16.00
24.25
9.19
401/07 INH (C)
17.15
14.93
9.85
6.96
9.19
27.86
401/08 INH (C)
4.16
7.80
11.31
8.57
2.29
2.64
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
CONCLUSIONS
•
•
•
•
•
•
•
THIORIDAZINE HAS IN VITRO ACTIVITY AGAINST ALL STRAINS OF Mtb.
THIORIDAZINE ENHANCES INTRACELLULAR KILLING OF MDR/XDR Mtb.
THIORIDAZINE CURES THE MOUSE OF AN MDR Mtb INFECTION.
THIORIDAZINE CURES THE HUMAN OF AN XDR-TB INFECTION.
DUAL MECHANISM OF ACTION:
A) INHIBITION OF K+ EFFLUX OF MACROPHAGE PERMITTING
ACIDIFICATION OF PHAGOLYSOSOME.
B) INHIBITION OF EFFLUX PUMPS OF Mtb THAT BESTOW MDR
PHENOTYPE.
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.
May 2011 Special Issue of PRI:
Thioridazine MDR/XDR TB
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.).
ISN
RESEARCH TEAM
L Amaral, May 201