- Email: [email protected]
Neuroleptic drugs in the treatment of tuberculosis: Minimal inhibitory concentrations of different phenothiazines against Mycobacterium tuberculosis
Accepted Manuscript Neuroleptic Drugs in the Treatment of Tuberculosis: Minimal Inhibitory Concentrations of Different Phenothiazines Against M. tuberculosis Dr. med. Silvan Vesenbeckh, MD MPH, Dr. med. David Krieger, Gudrun Bettermann, Dr. med. Nicolas Schönfeld, Dr. med. Torsten Thomas Bauer, Prof., Dr. med. Holger Rüssmann, Prof., Dr. med. Harald Mauch, Prof. PII:
S1472-9792(15)30292-4
DOI:
10.1016/j.tube.2016.02.003
Reference:
YTUBE 1426
To appear in:
Tuberculosis
Received Date: 18 December 2015 Revised Date:
12 February 2016
Accepted Date: 13 February 2016
Please cite this article as: Vesenbeckh S, Krieger D, Bettermann G, Schönfeld N, Bauer TT, Rüssmann H, Mauch H, Neuroleptic Drugs in the Treatment of Tuberculosis: Minimal Inhibitory Concentrations of Different Phenothiazines Against M. tuberculosis, Tuberculosis (2016), doi: 10.1016/j.tube.2016.02.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Neuroleptic Drugs in the Treatment of Tuberculosis: Minimal Inhibitory
2
Concentrations of Different Phenothiazines Against M. tuberculosis Authors:
5
Dr. med. Silvan VESENBECKH (a) [email protected]
6
Dr. med. David KRIEGER (a) [email protected]
7
Gudrun BETTERMANN (b) [email protected]
8
Dr. med. Nicolas SCHÖNFELD (a) [email protected]
9
Prof. Dr. med. Torsten Thomas BAUER (a+c) [email protected]
SC
4
Prof. Dr. med. Holger RÜSSMANN (b) [email protected]
11
Prof. Dr. med. Harald MAUCH (b) [email protected]
13
(a)
Department of Pneumology, Lungenklinik Heckeshorn, HELIOS Klinikum Emil von Behring,
14
Walterhöfer Str 11, 14165 Berlin, Germany
15
(b)
16
Germany
17
(c)
18
14165 Berlin, Germany
Institute of Microbiology, HELIOS Klinikum Emil von Behring, Walterhöfer Str 11, 14165 Berlin,
Deutsches Zentralkomitee zur Bekämpfung von Tuberkulose (DZK), Building Q, Walterhöfer Str 11,
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Corresponding author: Silvan Vesenbeckh MD MPH, HELIOS Klinikum Emil von Behring,
21
Walterhöferstr. 11, 14165 Berlin, Germany, Telephone: +49-30-8102-1875, Fax: +49-30-8102-42778,
22
email: [email protected]
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ACCEPTED MANUSCRIPT Summary
25
Due to an increase of drug resistant TB, alternative drugs that are not currently listed in the
26
WHO guidelines on MDR TB treatment are currently being evaluated. Our group tested 100
27
susceptible, 20 MDR and 2 XDR Mtb strains against the phenothiazine derivatives thioridazine,
28
trifluoperazine and triflupromazine. MIC testing was performed on Middlebrook 7H10 agar and
29
was defined as the lowest drug concentration that inhibits ≥ 99% of the bacterial population. We
30
confirm very good in vitro activity of phenothiazines against M. tuberculosis. In > 77% of all
31
strains MICs of ≤10 µg/ml were found.
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24
32
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2
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Key Words
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Public Health; drug development; guideline; antibiotic resistance; efflux pump
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3
ACCEPTED MANUSCRIPT 1. Introduction
38
With interest we are following the current discussion about innovating multidrug-resistant (MDR)
39
tuberculosis (TB) therapy by expanding the indication of otherwise established non-TB drugs to the
40
treatment of TB 1. This idea is compelling because new drug discoveries probably cannot keep up with
41
the emergence of antibiotic resistances and the pricing scheme of the most recent drugs on the market,
42
delamanid and bedaquiline, keeps them out of reach for the countries most in need 2. Over the past
43
years, a series of well-characterized affordable and safe drugs, including phenothiazines, were shown to
44
have in vitro and in vivo biological activity against tuberculosis 1. Phenothiazines mainly act through
45
inhibition of the bacterial efflux pump so that the drug can be concentrated within the bacterial cell 3.
46
According to Crowle et al. phenothiazines are accumulated up to 100-fold within macrophages that
47
contain M. tuberculosis (Mtb) 4, leading to concentrations that kill intracellular MDR-Mtb strains
48
without, however, being toxic to the macrophage 5, 6. In autopsy studies of human lungs concentrations
49
up to 300 times higher were found when compared to drug concentrations in interstitial fluids 7. Studies
50
in mice suggest that thioridazine is effective against susceptible and multidrug-resistant Mtb in vivo
51
and in Argentina and India, thioridazine was already used in the salvage therapy of extensively drug
52
resistant (XDR) TB in humans, with encouraging, but preliminary results
53
investigated thioridazine. The aim of our study was to evaluate and compare the in vitro anti-TB
54
activity of other phenothiazine derivatives than thioridazine.
SC
M AN U
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. The majority of studies
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9, 10
8
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55
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4
ACCEPTED MANUSCRIPT 2. Material and Methods
58
For decades, our laboratory routinely determines minimal inhibitory concentrations (MIC) of all first
59
and most second line TB drugs listed in the WHO guidelines. In order to support the body of evidence
60
for a potential additional role of phenothiazines, we tested the phenothiazine derivatives thioridazine,
61
trifluoperazine and triflupromazine against a panel of 100 susceptible Mtb strains and 22 multidrug
62
resistant strains, of which two were also extensively drug resistant. All strains are originated from
63
patients with eastern European provenance and were collected during clinical routine procedures at our
64
centre between 2002 and 2014. The strains were culturally determined to be Mtb, genotyping was not
65
performed routinely, but clinical copy strains were not included.
SC
66
RI PT
57
67
MIC testing was performed on Middlebrook 7H10 agar using the proportion method as described
68
elsewhere
69
bacterial population in comparison with the drug free control plate after 28 days of incubation. No
70
ethical approval was required for this retrospective study.
. MIC99 was defined as the lowest drug concentration that inhibits at least 99% of the
M AN U
11
71
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ACCEPTED MANUSCRIPT 3. Results
74
Some of the strains failed to regrow so that not all 100 Mtb strains could be evaluated for all three
75
drugs. Of the susceptible strains, 93 could be tested against thioridazine, 96 against trifluoperazine and
76
70 against triflupromazine. For thioridazine, MICs were 5 µg/ml in 14/93 samples (15%), 10 µg/ml in
77
58/93 samples (62%) and 20 µg/ml in 21/93 samples (23%). MICs in the trifluoperazine group were at
78
5 µg/ml in 5 samples (5%), at 10 µg/ml in 69 samples (72%) and at 20 µg/ml in 22 samples (23%). Out
79
of 70 samples tested against triflupromazine a MIC of 5, 10 and 20 µg/ml was found in 8 cases (11%),
80
32 cases (46%) and 30 cases (43%), respectively (table 1).
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81
All of the MDR and XDR strains could be evaluated, they revealed a MIC of 10 µg/ml in all strains
83
tested against thioridazine and in 95% of all strains tested against trifluoperazine. In the latter group,
84
one strain (XDR) revealed a MIC of 5 µg/ml (5%). In the triflupromazine group, 19 strains showed a
85
MIC of 10 µg/ml (86%) and 3 strains of 20 µg/ml (14%) (table 1).
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ACCEPTED MANUSCRIPT 4. Discussion
90
Phenothiazines can be classified into three major groups according to their substituent on nitrogen. The
91
so far best studied and most promising candidate with the best risk-benefit profile seems to be
92
thioridazine, but other chemically modified derivatives were successfully investigated in the past12. We
93
tested one derivative out of each of the above-mentioned groups. All tested derivatives are readily
94
available drugs.
RI PT
89
95
Our susceptibility testing confirms in vitro activity of the three tested phenothiazine derivatives against
97
Mtb, especially for thioridazine and trifluoperazine. A MIC99 of ≤10 µg/ml was found in > 77% of all
98
tested strains. The MICs for susceptible and drug resistant Mtb strains were found to be very similar,
99
varying by ±1 dilution only. These results are in line with the literature where MIC50 values between 2.5 and 7.5 µg/ml were reported for trifluoperazine 13 and 2.5 µg/ml for thioridazine 12.
M AN U
100
SC
96
101
There is evidence in the literature to suggest that even low dose phenothiazines can lead to effective
103
concentrations in vivo. In a 1996 pharmacokinetic study in humans mean peak plasma levels of 278
104
nmol/l (≈ 0.1 µg/ml) were found 2-3 hours after oral administration of 50 mg thioridazine 6. As shown
105
by Crowle et al. up to 100-fold higher intracellular concentrations are achieved in situ through
106
accumulation of phenothiazines within macrophages that contain Mtb 4. This might be the explanation
107
why Ordway et al. reported complete killing of susceptible and MDR strains within macrophages after
108
3 days of incubation with thioridazine at only 0.1 µg/ml 5, whereas a minimum bactericidal
109
concentration (MBC) between 30 and 50 µg/ml was found when macrophages were absent 5.
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Our data suggest, that phenothiazines are promising candidates for adjuvant TB treatment.
112
Phenothiazine derivatives have the potential to increase the effectiveness of other anti-TB drugs when
113
added to the standard multidrug regimens in MDR TB
114
streptomycin against polydrug resistant Mtb were shown to be enhanced
115
mice equally showed significant synergistic effects when thioridazine was added as an adjuvant to a
116
regimen consisting of rifampicin, isoniazid and pyrazinamide 8. Due to the proposed mechanism of
117
action (i.e. the inhibition of the efflux pump), first line TB drugs to which resistances have developed
118
could potentially regain effectivity when phenothiazines are administered as adjuvants alongside those
119
drugs. Besides, the modes of action of phenothiazines explain why the occurrence of resistances against
120
phenothiazines is unlikely
121
dormant drug resistant strains of Mtb 13, 15.
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3
14
. In vitro activities of rifampicin and 4, 14
, and in vivo studies in
and this group of drugs might even be active against non-replicating,
7
ACCEPTED MANUSCRIPT 122 Last but not least, phenothiazines are prescribed as antipsychotic drugs for more than 60 years and
124
clinical complications are well characterized as compared with drugs that recently hit the market.
125
Thioridazine can be considered a safe drug when administered thoughtfully and for periods no longer
126
than 2-3 months. In addition, much lower drug levels would be needed in the treatment of TB
127
compared to the treatment of psychosis 3. The most common side effects include drowsiness and
128
transient mild retinopathy 3. However, cardiac side effects with prolonged QT interval, arrhythmias and
129
sudden death in rare circumstances have also been reported when used as antipsychotic drug (10-15
130
deaths per 10,000 person-years) and must be monitored carefully
131
thioridazine was shown to be structurally specific to this particular phenothiazine, its antimicrobial
132
properties are shared by many other phenothiazine derivatives.
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. While cardiotoxicity of
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ACCEPTED MANUSCRIPT 5. Conclusions
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The current increase in resistant TB is not met by a similar increase in affordable treatment options. We
139
believe that well-known drugs with anti-mycobacterial activity that are not currently listed in the WHO
140
guidelines on MDR TB treatment should be considered as alternative TB drugs. Phenothiazines
141
constitute such alternative candidates. Our in vitro testing suggests that different derivatives of this
142
drug are equally effective against tuberculosis. It remains to be investigated whether further chemical
143
modifications of the phenothiazine group might lead to phenothiazine enantiomers with a more
144
favourable adverse reaction profile and even more suitable antimicrobial activity.
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Role of the funding source
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The authors did not receive external funding for this study and declare no conflict of interests.
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References
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JW. Potential antimicrobial agents for the treatment of multidrug-resistant tuberculosis. Eur Respir J
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2014;43:884-897. doi: 10.1183/09031936.00113713
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2.
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implications. Expert Opin Investig Drugs 2011;20:1665-1676. doi: 10.1517/13543784.2011.628657
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mycobacterial infections. Chemotherapy 1992;38:410-419.
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Molnar J, Amaral L. Clinical concentrations of thioridazine kill intracellular multidrug-resistant
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Mycobacterium tuberculosis. Antimicrob Agents Chemother 2003;47:917-922.
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pharmacodynamic effects of thioridazine and its metabolites in humans. Clin Pharmacol Ther
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tissues during early-stages postmortem. Journal of forensic sciences 1999;44:10-16.
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Ingen J, Boeree MJ. The antipsychotic thioridazine shows promising therapeutic activity in a mouse
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model of multidrug-resistant tuberculosis. PLoS One 2010;5 doi: 10.1371/journal.pone.0012640
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extensively drug-resistant tuberculosis (XDR-TB) and the need for global trials is now! Int J
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Antimicrob Agents 2010;35:524-526. doi: 10.1016/j.ijantimicag.2009.12.019
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Alsaad N, Wilffert B, van Altena R, de Lange WC, van der Werf TS, Kosterink JG, Alffenaar
Burki T. Improving the health of the tuberculosis drug pipeline. Lancet Infect Dis 2014;14:102-
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Sharma S, Singh A. Phenothiazines as anti-tubercular agents: mechanistic insights and clinical
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Crowle AJ, Douvas GS, May MH. Chlorpromazine: a drug potentially useful for treating
Ordway D, Viveiros M, Leandro C, Bettencourt R, Almeida J, Martins M, Kristiansen JE,
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Hartigan-Go K, Bateman DN, Nyberg G, Martensson E, Thomas SH. Concentration-related
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Moriya F, Hashimoto Y. Redistribution of basic drugs into cardiac blood from surrounding
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van Soolingen D, Hernandez-Pando R, Orozco H, Aguilar D, Magis-Escurra C, Amaral L, van
Amaral L, Boeree MJ, Gillespie SH, Udwadia ZF, van Soolingen D. Thioridazine cures
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Ritacco V, van Soolingen D. Successful alternative treatment of extensively drug-resistant tuberculosis
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in Argentina with a combination of linezolid, moxifloxacin and thioridazine. J Antimicrob Chemother
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2012;67:473-477. doi: 10.1093/jac/dkr500
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Minimal inhibitory concentrations of first-line drugs of multidrug-resistant tuberculosis isolates. Lung
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India 2012;29:309-312. doi: 10.4103/0970-2113.102794
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E, Amaral L. In vitro and ex vivo activity of thioridazine derivatives against Mycobacterium
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tuberculosis. Int J Antimicrob Agents 2007;29:338-340. doi: 10.1016/j.ijantimicag.2006.10.013
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replicating
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10.1371/journal.pone.0044245
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Mycobacterium tuberculosis by phenothiazines. Int J Antimicrob Agents 2001;17:225-228.
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model of latent tuberculosis. Tuberculosis 2014;94:695-700. doi: 10.1016/j.tube.2014.08.016
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and sudden death. The American journal of psychiatry 2001;158:1774-1782.
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Schonfeld N, Bergmann T, Vesenbeckh S, Mauch H, Bettermann G, Bauer TT, Russmann H.
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Martins M, Schelz Z, Martins A, Molnar J, Hajos G, Riedl Z, Viveiros M, Yalcin I, Aki-Sener
Advani MJ, Siddiqui I, Sharma P, Reddy H. Activity of trifluoperazine against replicating, nonand
drug
resistant
M.
tuberculosis.
PLoS
One
2012;7:e44245.
doi:
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Viveiros M, Amaral L. Enhancement of antibiotic activity against poly-drug resistant
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Singh A, Sharma S. Chemotherapeutic efficacy of thioridazine as an adjunct drug in a murine
Glassman AH, Bigger JT, Jr. Antipsychotic drugs: prolonged QTc interval, torsade de pointes,
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Abbate E, Vescovo M, Natiello M, Cufre M, Garcia A, Gonzalez Montaner P, Ambroggi M,
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Table 1: Minimal Inhibitory Concentrations (MIC) of different phenothiazine derivatives against
197
susceptible and resistant M. tuberculosis (Mtb) strains (MDR – multidrug-resistant, XDR – extensively
198
resistant).
199
Triflupromazine
n
5
10
20
MDR/XDR
22
0
22 (100%)
0
Susceptible
93
14 (15%)
58 (62%)
21 (23%)
MDR/XDR
22
1 (5%)
21 (95%)
0
Susceptible
96
5 (5%)
69 (72%)
22 (23%)
MDR/XDR
22
0
19 (86%)
3 (14%)
Susceptible
70
8 (11%)
32 (46%)
30 (43%)
200
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Trifluoperazine
Mtb strain
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MIC (µg/ml)
13
S1472-9792(15)30292-4
DOI:
10.1016/j.tube.2016.02.003
Reference:
YTUBE 1426
To appear in:
Tuberculosis
Received Date: 18 December 2015 Revised Date:
12 February 2016
Accepted Date: 13 February 2016
Please cite this article as: Vesenbeckh S, Krieger D, Bettermann G, Schönfeld N, Bauer TT, Rüssmann H, Mauch H, Neuroleptic Drugs in the Treatment of Tuberculosis: Minimal Inhibitory Concentrations of Different Phenothiazines Against M. tuberculosis, Tuberculosis (2016), doi: 10.1016/j.tube.2016.02.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Neuroleptic Drugs in the Treatment of Tuberculosis: Minimal Inhibitory
2
Concentrations of Different Phenothiazines Against M. tuberculosis Authors:
5
Dr. med. Silvan VESENBECKH (a) [email protected]
6
Dr. med. David KRIEGER (a) [email protected]
7
Gudrun BETTERMANN (b) [email protected]
8
Dr. med. Nicolas SCHÖNFELD (a) [email protected]
9
Prof. Dr. med. Torsten Thomas BAUER (a+c) [email protected]
SC
4
Prof. Dr. med. Holger RÜSSMANN (b) [email protected]
11
Prof. Dr. med. Harald MAUCH (b) [email protected]
13
(a)
Department of Pneumology, Lungenklinik Heckeshorn, HELIOS Klinikum Emil von Behring,
14
Walterhöfer Str 11, 14165 Berlin, Germany
15
(b)
16
Germany
17
(c)
18
14165 Berlin, Germany
Institute of Microbiology, HELIOS Klinikum Emil von Behring, Walterhöfer Str 11, 14165 Berlin,
Deutsches Zentralkomitee zur Bekämpfung von Tuberkulose (DZK), Building Q, Walterhöfer Str 11,
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12
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Corresponding author: Silvan Vesenbeckh MD MPH, HELIOS Klinikum Emil von Behring,
21
Walterhöferstr. 11, 14165 Berlin, Germany, Telephone: +49-30-8102-1875, Fax: +49-30-8102-42778,
22
email: [email protected]
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EP
20
1
ACCEPTED MANUSCRIPT Summary
25
Due to an increase of drug resistant TB, alternative drugs that are not currently listed in the
26
WHO guidelines on MDR TB treatment are currently being evaluated. Our group tested 100
27
susceptible, 20 MDR and 2 XDR Mtb strains against the phenothiazine derivatives thioridazine,
28
trifluoperazine and triflupromazine. MIC testing was performed on Middlebrook 7H10 agar and
29
was defined as the lowest drug concentration that inhibits ≥ 99% of the bacterial population. We
30
confirm very good in vitro activity of phenothiazines against M. tuberculosis. In > 77% of all
31
strains MICs of ≤10 µg/ml were found.
RI PT
24
32
AC C
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M AN U
SC
33
2
ACCEPTED MANUSCRIPT 34
Key Words
35
Public Health; drug development; guideline; antibiotic resistance; efflux pump
AC C
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M AN U
SC
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36
3
ACCEPTED MANUSCRIPT 1. Introduction
38
With interest we are following the current discussion about innovating multidrug-resistant (MDR)
39
tuberculosis (TB) therapy by expanding the indication of otherwise established non-TB drugs to the
40
treatment of TB 1. This idea is compelling because new drug discoveries probably cannot keep up with
41
the emergence of antibiotic resistances and the pricing scheme of the most recent drugs on the market,
42
delamanid and bedaquiline, keeps them out of reach for the countries most in need 2. Over the past
43
years, a series of well-characterized affordable and safe drugs, including phenothiazines, were shown to
44
have in vitro and in vivo biological activity against tuberculosis 1. Phenothiazines mainly act through
45
inhibition of the bacterial efflux pump so that the drug can be concentrated within the bacterial cell 3.
46
According to Crowle et al. phenothiazines are accumulated up to 100-fold within macrophages that
47
contain M. tuberculosis (Mtb) 4, leading to concentrations that kill intracellular MDR-Mtb strains
48
without, however, being toxic to the macrophage 5, 6. In autopsy studies of human lungs concentrations
49
up to 300 times higher were found when compared to drug concentrations in interstitial fluids 7. Studies
50
in mice suggest that thioridazine is effective against susceptible and multidrug-resistant Mtb in vivo
51
and in Argentina and India, thioridazine was already used in the salvage therapy of extensively drug
52
resistant (XDR) TB in humans, with encouraging, but preliminary results
53
investigated thioridazine. The aim of our study was to evaluate and compare the in vitro anti-TB
54
activity of other phenothiazine derivatives than thioridazine.
SC
M AN U
TE D
. The majority of studies
EP
56
9, 10
8
AC C
55
RI PT
37
4
ACCEPTED MANUSCRIPT 2. Material and Methods
58
For decades, our laboratory routinely determines minimal inhibitory concentrations (MIC) of all first
59
and most second line TB drugs listed in the WHO guidelines. In order to support the body of evidence
60
for a potential additional role of phenothiazines, we tested the phenothiazine derivatives thioridazine,
61
trifluoperazine and triflupromazine against a panel of 100 susceptible Mtb strains and 22 multidrug
62
resistant strains, of which two were also extensively drug resistant. All strains are originated from
63
patients with eastern European provenance and were collected during clinical routine procedures at our
64
centre between 2002 and 2014. The strains were culturally determined to be Mtb, genotyping was not
65
performed routinely, but clinical copy strains were not included.
SC
66
RI PT
57
67
MIC testing was performed on Middlebrook 7H10 agar using the proportion method as described
68
elsewhere
69
bacterial population in comparison with the drug free control plate after 28 days of incubation. No
70
ethical approval was required for this retrospective study.
. MIC99 was defined as the lowest drug concentration that inhibits at least 99% of the
M AN U
11
71
AC C
EP
TE D
72
5
ACCEPTED MANUSCRIPT 3. Results
74
Some of the strains failed to regrow so that not all 100 Mtb strains could be evaluated for all three
75
drugs. Of the susceptible strains, 93 could be tested against thioridazine, 96 against trifluoperazine and
76
70 against triflupromazine. For thioridazine, MICs were 5 µg/ml in 14/93 samples (15%), 10 µg/ml in
77
58/93 samples (62%) and 20 µg/ml in 21/93 samples (23%). MICs in the trifluoperazine group were at
78
5 µg/ml in 5 samples (5%), at 10 µg/ml in 69 samples (72%) and at 20 µg/ml in 22 samples (23%). Out
79
of 70 samples tested against triflupromazine a MIC of 5, 10 and 20 µg/ml was found in 8 cases (11%),
80
32 cases (46%) and 30 cases (43%), respectively (table 1).
RI PT
73
81
All of the MDR and XDR strains could be evaluated, they revealed a MIC of 10 µg/ml in all strains
83
tested against thioridazine and in 95% of all strains tested against trifluoperazine. In the latter group,
84
one strain (XDR) revealed a MIC of 5 µg/ml (5%). In the triflupromazine group, 19 strains showed a
85
MIC of 10 µg/ml (86%) and 3 strains of 20 µg/ml (14%) (table 1).
M AN U
SC
82
86 87
AC C
EP
TE D
88
6
ACCEPTED MANUSCRIPT 4. Discussion
90
Phenothiazines can be classified into three major groups according to their substituent on nitrogen. The
91
so far best studied and most promising candidate with the best risk-benefit profile seems to be
92
thioridazine, but other chemically modified derivatives were successfully investigated in the past12. We
93
tested one derivative out of each of the above-mentioned groups. All tested derivatives are readily
94
available drugs.
RI PT
89
95
Our susceptibility testing confirms in vitro activity of the three tested phenothiazine derivatives against
97
Mtb, especially for thioridazine and trifluoperazine. A MIC99 of ≤10 µg/ml was found in > 77% of all
98
tested strains. The MICs for susceptible and drug resistant Mtb strains were found to be very similar,
99
varying by ±1 dilution only. These results are in line with the literature where MIC50 values between 2.5 and 7.5 µg/ml were reported for trifluoperazine 13 and 2.5 µg/ml for thioridazine 12.
M AN U
100
SC
96
101
There is evidence in the literature to suggest that even low dose phenothiazines can lead to effective
103
concentrations in vivo. In a 1996 pharmacokinetic study in humans mean peak plasma levels of 278
104
nmol/l (≈ 0.1 µg/ml) were found 2-3 hours after oral administration of 50 mg thioridazine 6. As shown
105
by Crowle et al. up to 100-fold higher intracellular concentrations are achieved in situ through
106
accumulation of phenothiazines within macrophages that contain Mtb 4. This might be the explanation
107
why Ordway et al. reported complete killing of susceptible and MDR strains within macrophages after
108
3 days of incubation with thioridazine at only 0.1 µg/ml 5, whereas a minimum bactericidal
109
concentration (MBC) between 30 and 50 µg/ml was found when macrophages were absent 5.
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Our data suggest, that phenothiazines are promising candidates for adjuvant TB treatment.
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Phenothiazine derivatives have the potential to increase the effectiveness of other anti-TB drugs when
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added to the standard multidrug regimens in MDR TB
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streptomycin against polydrug resistant Mtb were shown to be enhanced
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mice equally showed significant synergistic effects when thioridazine was added as an adjuvant to a
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regimen consisting of rifampicin, isoniazid and pyrazinamide 8. Due to the proposed mechanism of
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action (i.e. the inhibition of the efflux pump), first line TB drugs to which resistances have developed
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could potentially regain effectivity when phenothiazines are administered as adjuvants alongside those
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drugs. Besides, the modes of action of phenothiazines explain why the occurrence of resistances against
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phenothiazines is unlikely
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dormant drug resistant strains of Mtb 13, 15.
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3
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. In vitro activities of rifampicin and 4, 14
, and in vivo studies in
and this group of drugs might even be active against non-replicating,
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ACCEPTED MANUSCRIPT 122 Last but not least, phenothiazines are prescribed as antipsychotic drugs for more than 60 years and
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clinical complications are well characterized as compared with drugs that recently hit the market.
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Thioridazine can be considered a safe drug when administered thoughtfully and for periods no longer
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than 2-3 months. In addition, much lower drug levels would be needed in the treatment of TB
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compared to the treatment of psychosis 3. The most common side effects include drowsiness and
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transient mild retinopathy 3. However, cardiac side effects with prolonged QT interval, arrhythmias and
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sudden death in rare circumstances have also been reported when used as antipsychotic drug (10-15
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deaths per 10,000 person-years) and must be monitored carefully
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thioridazine was shown to be structurally specific to this particular phenothiazine, its antimicrobial
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properties are shared by many other phenothiazine derivatives.
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. While cardiotoxicity of
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The current increase in resistant TB is not met by a similar increase in affordable treatment options. We
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believe that well-known drugs with anti-mycobacterial activity that are not currently listed in the WHO
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guidelines on MDR TB treatment should be considered as alternative TB drugs. Phenothiazines
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constitute such alternative candidates. Our in vitro testing suggests that different derivatives of this
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drug are equally effective against tuberculosis. It remains to be investigated whether further chemical
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modifications of the phenothiazine group might lead to phenothiazine enantiomers with a more
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favourable adverse reaction profile and even more suitable antimicrobial activity.
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Role of the funding source
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The authors did not receive external funding for this study and declare no conflict of interests.
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ACCEPTED MANUSCRIPT 150 151 152
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Table 1: Minimal Inhibitory Concentrations (MIC) of different phenothiazine derivatives against
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susceptible and resistant M. tuberculosis (Mtb) strains (MDR – multidrug-resistant, XDR – extensively
198
resistant).
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Triflupromazine
n
5
10
20
MDR/XDR
22
0
22 (100%)
0
Susceptible
93
14 (15%)
58 (62%)
21 (23%)
MDR/XDR
22
1 (5%)
21 (95%)
0
Susceptible
96
5 (5%)
69 (72%)
22 (23%)
MDR/XDR
22
0
19 (86%)
3 (14%)
Susceptible
70
8 (11%)
32 (46%)
30 (43%)
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Trifluoperazine
Mtb strain
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Thioridazine
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MIC (µg/ml)
13