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The Mechanism by which the Phenothiazine Thioridazine Contributes to Cure Problematic Drug-Resistant Forms of Pulmonary Tuberculosis: Recent Patents for “New Use” Leonard Amaral1*, Ana Martins2,3, Gabriella Spengler2, Attila Hunyadi4 and Joseph Molnar2 1

Center for Malaria and Other Tropical Diseases (CMDT), Institute of Hygiene and Tropical Medicine, Universidade Nova de Lisboa, Rua da Junqueira 100, Lisbon 1349-008, Portugal; 2Department of Medical Microbiology and Immunobiology, University of Szeged, Dóm tér 9, Szeged 6720, Hungary; 3Unidade de Parasitologia e Microbiologia Médica, Institute of Hygiene and Tropical Medicine, Universidade Nova de Lisboa, Lisbon, Portugal, Rua da Junqueira 100, Lisbon 1349-008, Portugal; 4Institute of Pharmacognosy, Faculty of Pharmacy, University of Szeged, Eötvös u. 6, 6720 Szeged, Hungary Received: November 6, 2013; Revised: December 6, 2013; Accepted: December 7, 2013

Abstract: At this moment, over half million patients suffer from multi-drug resistant tuberculosis (MDR-TB) according to the data from the WHO. A large majority is terminally ill with essentially incurable pulmonary tuberculosis. This herein mini-review provides the experimental and observational evidence that a specific phenothiazine, thioridazine, will contribute to cure any form of drug-resistant tuberculosis. This antipsychotic agent is no longer under patent protection for its initial use. The reader is informed on the recent developments in patenting this compound for “new use” with a special emphasis on the aspects of drug-resistance. Given that economic motivation can stimulate the use of this drug as an antitubercular agent, future prospects are also discussed.

Keywords: Curing of XDR TB, mechanism of killing, mycobacterium tuberculosis, thioridazine. INTRODUCTION The frequency of drug resistance among Mycobacterium tuberculosis infections [1-6] and its progression to multidrug resistance (MDR-TB) [3,7-13] and beyond (extensively drug resistance (XDR-TB) [14-21] and the ill-defined total drug resistance (TDR-TB) [21-27]) continue to increase. Generally, the development and progression of bacterial resistance is primarily due to continued ineffective therapy that results in the selection of antibiotic resistant bacteria in patients within a health care facility [13,15]. In addition, drug resistant strains are frequently transmitted within the hospital setting (nosocomial) [15] and the community [8], which, when further treated ineffectively, lead to higher levels of resistance [28-33]. Ineffective therapy involves inappropriate dosage or prescription of antibiotics [34-38], low exposure or non-compliance by the patient [39-42]. The emergence of antibiotic resistant tuberculosis is an actual problem. Taking into consideration that, in the past three decades, only a few safe and effective anti-tuberculosis agents were developed, the increasing number of MDR, XDR and TDR-TB cases are a significant threat to indigent countries as well as to the affluent countries of the globe. Consequently, new antimicrobial agents of alternative classes and from a wide variety of sources (including natural sources such as the microbial *Address correspondence to this author at the Travel Medicine, Centro de Malária Doenças Tropicais, Institute of Hygiene and Tropical Medicine, Universidade Nova de Lisboa, Rua Junqueira 100, 1349-008 Lisbon, Portugal; Tel: +351213652600; Fax: +351213632105; E-mail: [email protected] 1574-891X/13 $100.00+.00

community, animals and plants, chemical modifications of existing antimicrobial agents and de novo syntheses) have been the focus of intensive research that involved not only anti-tuberculosis agents but also agents against resistant bacterial infections in general. In this mini-review we will focus on the possible use of phenothiazines, agents that are usually employed for non-infectious pathologies. This chemical group has for many years been known to possess a wideranging antimicrobial activity, and has recently been subjected to patenting with “new uses”. In addition, further safe and effective phenothiazine derivatives are also going to be introduced – due to their plethora of mechanisms, emerging resistance to these agents is not anticipated. PHENOTHIAZINES AND TUBERCULOSIS Phenothiazines are heterocyclic compounds from which a great number of medicinal compounds have been derived during the last century, for a broad range of different indications including antimalarial, antihistamic, antipsychotic, antiparkinson and antiemetic use. A. Bernthsen produced the first synthetic phenothiazine in 1883 [43] by the reaction of diarylamine with sulfur. However, the first patent for a phenothiazine dye (methylene blue) was issued earlier in December 15, 1877 to the German company BASF [44]. Later on, the synthesis of mauve dyes by Perkin led to an increase in popularity of dyestuffs and in turn encouraged research on the use of aniline derivatives as dye precursors [44]. The fact that such dye derivatives could be used to stain tissues for microscopy was soon realized. Scientists such as Robert © 2013 Bentham Science Publishers

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Koch and Paul Ehrlich showed that different cell types stained selectively with different dyes due to variances in their chemical structure. Because these dyes could be used as vital stains (staining of living cells), they were soon shown to affect physiological processes, such as the motility of bacteria and protozoa. This encouraged Guttmann and Ehrlich in 1891 to attempt, with success, to treat malaria with methylene blue [45]. Because methylene blue would stain the tissues of the patient receiving this dye, its use was never seriously considered for therapy of malaria. Nevertheless, Roehl, a student of Erhlich’s, continued to work with methylene blue and Plasmodium spp. (organisms that cause malaria) [46], and later, his research was also followed by Schulemann who successfully utilized a phenothiazinium salt for curing malaria in 1932 [47]. However, the phenothiazinium salts were still blue dyes, and this produced a bluish coloration of the skin in the patient. However, because in 1899 the administration of methylene blue to an animal was shown to produce lethargy [48], interest in methylene blue continued, not as a potential antimicrobial agent, but as a possible source for anti-psychotic agents. This became the focus of the biological properties of phenothiazines for the next six decades which eventually resulted in the first neuroleptic chlorpromazine (CPZ), a colorless phenothiazine [49]. The side effects observed during extensive global use of CPZ, suggested that CPZ could also serve as an antimicrobial agent. However, because this era (early 1960’s) was also the “Golden Age of Antibiotics”, there was no interest in the antimicrobial use of CPZ. It was the advent of multidrug resistance of tuberculosis infections during the 1990’s that promoted interest in the earlier studies that showed that CPZ could cure tuberculosis [50-60]. However, because all in vitro studies demonstrated that the concentration of CPZ required for the inhibition of the growth of Mycobacterium tuberculosis was many fold higher than that which could be safely achieved in the patient [61, 62], the use of CPZ for therapy of multi-drug resistant infections was not seriously considered. Moreover, the question of why CPZ could cure patients presenting with tuberculosis at conventional doses employed for therapy of psychosis, was not considered until the studies of Crowle et al. were published in 1992 [63]. These studies showed that exposure of macrophages containing phagocytosed Mycobacterium tuberculosis to clinically reachable concentrations of CPZ resulted in the killing of the mycobacteria. However, because CPZ produces a large number of serious negative side effects [64], the possible use of CPZ for therapy of tuberculosis was still resisted. This situation remained until the studies of Amaral et al showed that thioridazine (TZ) was equipotent to CPZ in vitro [65] and could inhibit the growth of both antibiotic sensitive and MDR Mycobacterium tuberculosis (resistant to isoniazid and rifampicin, the two most effective anti-tuberculosis agents), as well as strains that were resistant to all of the five first line anti-tubercular drugs. TZ is a much milder, and relatively safe neuroleptic as compared to CPZ. And just like CPZ [63], TZ has also been shown to promote the killing of intracellular strains of Mycobacterium tuberculosis regardless of its antibiotic resistance, i.e. multidrug resistant (MDR) [66, 67] or extensively drug resistant (XDR) strains [68]. Although TZ has also been shown to cure mice infected with antibiotic susceptible [69] and multidrug resistant strains of Mycobacterium tuberculosis [70], as shown by a recent

Amaral et al.

study, and confirming what had been predicted earlier by Amaral et al. [65], it does not cure guinea pigs presenting with extracellular infections TZ [71]. Nevertheless, 18 patients infected with XDR-TB have been cured with a combination of TZ and antibiotics to which the strains were initially resistant [72]. Moreover, the in vitro ability of this compound to kill dormant Mycobacterium tuberculosis [73] suggests that TZ could also be used for therapy of latent tuberculosis, alone or as an adjuvant, in combination with existing anti-tuberculosis drugs [74]. Of relevance to these latter studies, Sarathy and co-authors have shown that bactericidal activity of TZ remained comparable in both growing and dormant (nutrient starved) M. tuberculosis [75]. The above cited in vitro studies [73] have support the attempt to employ TZ for monotherapy of essentially terminal patients presenting with XDR TB [76]. Monotherapy with TZ has been shown to improve the quality of life of all patients placed on this “salvage programme”. Within a short period of time (weeks), these patients regained their appetite and gained weight; their night sweats obviated or reduced in frequency; and, due to the neuroleptic activity of TZ, the patients were calmer. However, although TZ can cure XDRTB patients [71, 78], those placed on “salvage therapy” had severe pulmonary damage. Because TZ does not repair or promote repair of the damaged lung parenchyma, all of the patients eventually succumbed to the severity of pulmonary damage caused by the infecting XDR Mycobacterium tuberculosis strain. Nevertheless, because patients were monitored prior to and during therapy with TZ, no detectable cardiac problems such as increased Qt intervals were observed in this [76] or in other studies tghat employed TZ for therapy of XDR-TB [71]. The potential value of employing TZ for therapy of antibiotic resistant infections has received support from the medical press [77-79]. Thioridazine is not the only phenothiazine that has been recommended for therapy of pulmonary tuberculosis. In general, many phenothiazines have been implicated for antitubercular activity [62, 80-86]. Among these are trifluoperazine [87-94], methdilazine [95, 96], promazine [97, 98], promethazine [97, 98], fluphenazin [99], propiomazine [100], and the methylene blue related toluidine blue [101]. Figure 1 summarises the evolution of phenothiazine compounds derived from the parental methylene blue for therapy of pathologies unrelated to tuberculosis, but which compounds also possess antitubercular [44, 48] and/or antimalarial properties [44]. Moreover, derivatives made from any of the phenothiazines that have in vitro activity against Mycobacterium tuberculosis are also active [61, 67, 102, 103], suggesting ample opportunities for patenting of new analogs developed from known, active phenothiazines with even less side effects than those of TZ, as recently suggested by Musuka and co-authors [104]. It is important to mention, that the commercially available phenothiazines such as for example trifluoperazine, methdilazine, promazine, promethazine, fluphenazin and propiomazine are beyond patent protection as initially intended. Nevertheless, these compounds have been patented as adjuvants for the treatment of MDR cancer (patent expired in 2011) [105]; and, right afterwards, a new patent has been filed with a priority date of 28th March, 2012, claiming combination therapy of cancer with a chemotherapeutic agent and a dopamine receptor antagonist

Mechanism by which Thioridazine Enhances Killing of Intracellular

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(e.g. TZ), with a special emphasis on cancers stem cells (CSCs) [106]. Taking into account that intrinsic MDR is considered as one of the key properties of CSCs [107], the subject to be covered is indeed related. According to the MDR, XDR and TDR Mycobacterium tuberculosis, subjects of this herein paper, the initial step for actually reaching those in need has been made: a patent has been published in December, 2007, for the use of TZ and its derivatives for reversing anti-microbial drug resistance [108]. We must note, however, that, despite the six years passed since, we were unable to find any related clinical trials, which would certainly be of outmost importance and urgency in order to proceed towards an effective therapy of highly resistant mycobacterial infections. MECHANISM OF ACTION OF TZ: WHY IT CURES MULTI-DRUG, EXTENSIVELY DRUG RESISTANT AND PROBABLY TOTALLY DRUG RESISTANT TUBERCULOSIS Over-expressed efflux pumps of Mycobacterium tuberculosis render the organism multi-drug resistant [13]. Special attention has been given to those coded by the mmpL7, p55, efpA, mmr, Rv1258c and Rv2459 genes [109]. The activity of these efflux pumps can be suppressed by concentrations of TZ that have no effect on the viability of Mycobacterium tuberculosis rendering the organism susceptible to the antibiotic to which it was initially resistant as a consequence of the over-expression of its efflux pumps [109]. TZ has also been shown to inhibit the activity of the main efflux pumps of bacteria belonging to other species such as Escherichia

coli [110-115], Salmonella enterica serovar Typhimurium [116, 117], Enterobacter aerogenes [118], Staphylococcus aureus [119] as well as the ABCB1 efflux pump of cancer cells [120], suggesting that the activity of TZ against efflux pumps is quite broad. With respect to M. tuberculosis, the use of TZ has been suggested as an adjunct for therapy of MDR-TB infections whose MDR phenotype is due in part or completely to the presence of over-expressed efflux pumps [74, 121]. This suggestion has received further support from the studies of Dutta et al., who demonstrated that TZ has strong inhibitory activity against the genes that code for essential proteins of M. tuberculosis [122-124]. Consequently, we may conclude that the in vitro activity of TZ involves the inhibition of the efflux pumps of M. tuberculosis and that the in vitro exposure of this organism to TZ renders the organism susceptible to antibiotics to which it was initially resistant as a consequence of over-expressed efflux pumps [21]. Phenothiazines such as CPZ, TZ, trifluoperazine, etc., also inhibit the binding of calcium to calcium binding proteins such as calmodulin in eukaryotes [125], and interfere with other proteins involved in the regulation of cellular activity [126]. They inhibit the transport of calcium and potassium systems in eukaryotic cells [127-129] as well as in mycobacteria [89, 130] and E. coli [113]. In fact, in the later case, calcium was shown essential to the continuous activity of the thioridiazine sensitive efflux system [113]. The killing activity of the human macrophage as well as that of the neutrophil is dependent upon the retention of calcium and potassium within the phagolysosome of the cell [131]. These ions are needed for the activity of vacuolar ATPases, which in

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turn reduce the pH of the phagolysosome and, consequently, induce hydrolysis of the phagolysosome-entrapped bacterium [130]. Because agents such as oubain that inhibit transport of potassium, enhance the killing of intracellular M. tuberculosis [127], the mechanism by which TZ, an inhibitor of potassium transport, enhances the killing of intracellular M. tuberculosis is supposed to be due to the inhibition of the efflux of these ions from the phagolysosome of the nonkilling human macrophage, thereby activating its killing machinery [127]. This concept of enhancing the killing activity of the non-killing human macrophage of the alveolar structure of the lung is new and significant since it targets the macrophage [132]. Because targeting the intracellular M. tuberculosis with an antibiotic may be soon followed by a mutational response of the bacterium that renders it resistant to the antibiotic, the enhanced intracellular killing capacity of the macrophage promoted by TZ would bypass any mutational response by the organism. Moreover, if TZ is used as an adjunct with antibiotics that are effluxed by the intracellular M. tuberculosis, TZ will also inhibit the efflux of the antibiotic, rendering the organism susceptible. Furthermore, because macrophages are rich in lysosomes that concentrate this phenothiazine [133, 134], the concentration of intracellular TZ may reach levels associated with its minimal inhibitory concentration (MIC) or even its minimum bactericidal concentration [135-138] within the macrophage, without the need of giving those concentrations to the patient, which would otherwise be toxic.

conduct one or more well-designed, double-blind clinical trial(s), which, based on the previous experiences on patients treated with this drug, will most likely confirm the expected efficacy. Unfortunately, as of today, the patent protection of TZ and its derivatives as MDR reversal agents [108] does not appear to be utilized. NeverthelesFs, many other commercially available phenothiazines have in vitro activities as cited in this mini-review and the above mentioned patent focuses on TZ and its derivatives [108], consequentially not covering all prospective candidates. Considering this, several alternative choices are available for patenting under “new use”, which would allow a “fresh start” for the compound to be developed. However, the needed experimental proof that these phenothiazine agents have activity at the pulmonary macrophage of the alveolar unit (the site where the causative organism of pulmonary tuberculosis resides) is still absent.

Other studies also suggest that TZ, as well as other phenothiazine analogs, specifically inhibit respiratory chain related enzymes and affect key cellular mechanisms essential to the survival of M. tuberculosis under low-oxygen conditions (for example during intracellular infection) [84, 139]. Among those are; inhibition of NADH; menaquinone oxidoreductase activity [139], type II NADH-ubiquinone dehydrogenase [84] and the integral membrane succinate dehydrogenase [84], as well as inhibition of oxygen consumption and decrease of intracellular ATP levels of M. tuberculosis [84].

The authors acknowledge the Szeged Foundation for Cancer Research, the European Social Fund (TÁMOP 4.2.2/B-10/1-2010-0012 and TAMOP-4.2.2A-11/1/KONV2012-0035), and the Fundação para a Ciência e a Tecnologia, Portugal (PEsT-OE/SAU/UI0074/2011) for financial support. AM acknowledges the grant SFRH/BPD/81118/2011 provided by the FCT, Portugal.

Based upon the evidence presented, it is reasonable to expect that there are at least four mechanisms which together insure that TZ will be effective against any type of antibiotic resistant pulmonary tuberculosis infection; 1) enhanced killing activity of the human macrophage; 2) inhibition of the over-expressed efflux pump system of the antibiotic resistant Mycobacterium tuberculosis infecting the organism; 3) concentration of TZ to levels compatible with the MIC and MBC of the agent; and 4) inhibition of the oxygen consumption of the mycobacterium.

Consequently, we still urge the medical community to seriously consider thioridazine as an anti-tuberculotic agent, or, as a somewhat less preferable alternative, to conduct rapid development of another legislated phenothiazine drug for therapy of problematic tuberculosis infections. CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest. ACKNOWLEDGEMENTS

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