**5. Repositioning and combining drugs**

The combination of different drugs may pose the advantage of supra-additive effects, which may be synergistic, in parasite models such as *T. cruzi* [190], *Plasmodium falciparum*, *Trypanosoma rhodesiense* [191]. The identification of synergistic combinations is relevant since they tend to present higher selective indices [192, 193], consequently, avoiding side effects and potentially permitting development of antiparasitic agents used at lower concentrations.

The identification of drug combinations with multiple targets can lead to the use of novel multitarget mechanisms able to cope with the challenge of multigenic diseases [194] and/or chronic infections with complex pathophysiology. It is noteworthy that the pharmaceutical properties of the combination may be absent in the components alone [195], generating the innovative concept or science field termed polypharmacology with numerous applications on drug repurposing [196] and CD [197]. As the philosopher Aristotle (384–322 B.C.) stated: "The whole is greater than the sum of its parts."1

Furthermore, drug combinations are largely employed for preventing drug resistance [198–204]. However, this strategy is not constantly successful as the reports of resistance to the sulfadoxine-pyrimethamine combination began in the same year this antimalarial regimen entered the clinic [205]. Similarly, the discovery of artemisinin (ART) costed Youyou Tu over 30 years of hard work [206] and was worthy a Nobel Prize, but *P. falciparum* resistance to the drug was detected after about 10 years of use [207]. The antimalarial combination therapies based on the use of ART were considered key to the elimination of malaria [208], but in the very same year [209, 210] and even earlier [211], the arteminisin derivatives combination therapy failures were reported. In the case of CD, the problem may be even more upsetting as natural resistance isolates are arising, particularly in the Amazon region (*vide supra*). Thus, effective strategies to prevent different mechanisms of drug resistance to arise are immediately needed.

Approaching repositioned drugs with available pharmacokinetic and toxicological properties can shorten the long and expensive path between *in vitro* trials and new drugs. While the period between drug discovery and approval can be 12–16 years at a cost of US\$1–2 billion, repositioned drugs can enter the clinic in ½ the time, at *circa* 1/3 the cost [212], with much higher success rates [213].

Drug repositioning maybe a promising approach in CD [214–227]. Similarly, drug combinations may be instrumental in CD [197, 228–233], and both strategies may be employed and associated [214, 234–236]. Furthermore, drug combinations can

<sup>1</sup> "Since that which is compounded out of something so that the whole is one, not like a heap (…), then, is something-not only its elements (…) but also something else (…)" 'Metaphysics' Book VII by Aristotle, Translated by W. D. Ross, often misquoted or mistranslated.

increase success of drug repositioning [237]. In addition, it was accurately hypothesized that the combined use of repurposed drugs with BZ could be more efficacious than BZ alone [238].

### **5.1 Repositioning disulfiram**

Disulfiram (DS, 1,1′-disulfanediylbis(N,N-diethylmethanethioamide) also termed tetraethylthiuram disulfide; CAS no. 97-77-8; Molecular Formula: C10H20N2S4), a repositioned drug used in alcoholism and marketed as Antabuse® (**Figure 2**), was approved for medical use over 70 years ago and is widely used since then [239, 240].

At the very beginning, the discovery of thiocarbamates and its derivatives was serendipitous and showed clear signs of versatile perspectives that unequivocally culminated in the present promising repurposing strategies for both pharmaceutical and industrial applications [241, 242].

In the 1930s and 1940s, dithiocarbamates such as dimethyldithiocarbamates and diethyldithiocarbamates were used as pesticides against fungal pathogens on different crops [243], besides biocides in household products [244].

The industry plant physician E. E. Williams in 1937 observed that workers using tetramethylthiuram monosulfide and disulfide to facilitate the rubber vulcanization became alcohol-intolerant and quit consuming alcoholic beverages. The DSF-induced alcohol aversion was described in 1948 [245]. At that time, DSF was approached as a vermicide and employed as an ointment to treat scabies.

Afterward, besides alcoholism, DSF started to be studied for heavy metal poisoning, cancer [246–249], HIV [243, 250], as well as cocaine dependence, pathological gambling, and other psychiatric disorders [239] and other form of addiction, for example, the d-methamphetamine abuse [251]. Further tests are being performed focusing applications such as Alzheimer's disease [252], Lyme disease and babesiosis [253], tuberculosis [254], non-tuberculous mycobacteria infections [255], giardiasis [256], amoebiasis [257], obesity [258] and to revert drug resistance in different types of cancer [259–261], tuberculosis [262] bacterial infections [263], mycosis [264], giardiasis [265], etc. The repositioning of low-cost drugs such as DS is considered a "salvation" for global healthcare system [266].

Sodium diethylcarbamodithioate (**Figure 2**) (DETC also known as sodium (diethylcarbamothioyl)sulfanide; CAS no. 148-18-5; Molecular Formula: C5H11NS2.Na) is the first derivative of DSF, involved in many of the biological activities of the latter.

Seemingly DETC is less toxic than aspirin [243], widely used, and well tolerated in humans [267] for decades being used up to 800 mg/twice/week, with no adverse effects [268]. DETC also known as Imuthiol or Dithiocarb was used as immunomodulator with good results on AIDS patients [269, 270] and was clinically employed in chronic bronchitis, rheumatoid arthritis, tuberculosis, and chronic infection [271].

**Figure 2.** *Molecular structures of disulfiram (A) and sodium diethyldithiocarbamate (B).*

*Translational Research on Chagas Disease: Focusing on Drug Combination and Repositioning DOI: http://dx.doi.org/10.5772/intechopen.104231*

In a seminal report on its antiparasitic activity, DETC was demonstrated to be leishmanicidal [272]. Afterward, novel delivery systems were developed to optimize the leishmanicidal activity of DETC [273–275]. In this regard, novel drug delivery systems are also developed for DSF [276]. The data obtained on Leishmania amazonensis motivated us to move to CD, employing the repositioned drug DSF combined to the drug of first choice BZ. Tests on NFX are in progress.

It is worth remembering that CD pathophysiology is associated with oxidative stress (*vide supra*), and both DSF [277] and DETC [278] can act as antioxidants. In addition, modulation of oxidative stress comprises a valuable tool in heart disease therapeutics [279]. In addition, DSF has antimutagenic properties [280].

#### **5.2 Disulfiram combined to benznidazole in Chagas disease**

Both DSF and DETC have antiparasitic activity on *T. cruzi* [281, 282], but the effectivity was not pronounced.

In our study, the DSF-BZ combination is promising since the antagonism of SOD activity can enhance oxidative stress in cancer cells [249] and *T. cruzi* [283]. In this regard, the antitumor activity of NTX is enhanced by SOD1 inhibition mediated by tetrathiomolybdate [284]. Both in vitro and in vivo experimental data confirmed the present assumption [Almeida-Silva et al., in press]. The SOD inhibition as well as TSH reaction by DSF/DETC can promote the intracellular accumulation of ROS leading to parasite death (**Figure 3**).

CD etiological therapy is often associated to severe adverse effects caused by the highly toxic drugs (*vide supra*). In this sense, the present innovation involves the advantage of employing DSF/DETC with cytoprotective properties [243] in different cell types.

DSF/DETC have neuroprotective [285], hepatoprotective [277], and nephroprotective [286] and even radioprotective [287, 288] activity. These protective effects may be beneficial in the treatment of parasitic diseases, because in the treatment of experimental infection by *T. rhodesiense*, DSF has marked protective activity (disulfiram rescue) against the toxic effects of diaminodichloroplatin and preventing the death of the treated organism [289].

Thus, the development of low-toxicity therapies may be expected, as DSF may have a protective action against the toxic effects of drugs such as cyclophosphamide [290], ifosfamide [291], N-nitrosodimethylamine [292], isoniazid [293] and the toxicity of α-naphthylisothiocyanate [294], acetaminophen [295], pyrrolizidines [296], the lethal effects of hypoxia [297], ischemia [298], as well as lead [299], cadmium [300], mercury, and other heavy metals [301]. Thus, DSF combinations can enable the development of safe medicines. Regarding CD, the cardioprotective and antioxidant activities of DSF/DETC as well as atrial neuroprotection [302] are particularly desirable [303–306]. In addition, DSF is effective as prophylactics in experimental colitis [307].

As drug resistance limits the successful CD therapy, the *T. cruzi* PgP expression has a pivotal role [308]. Therefore, it is relevant in the present approach that DSF/DETC inhibit PgP [261, 309, 310], causing the BZ accumulation within the parasite cytoplasm, enhancing trypanocidal activity, potentially reversing resistance phenotypes, such as MDR+ (**Figure 3**). Interestingly, the ABCC proteins from *T. cruzi* are involved in thiol transport [311]. In view of the glutathione-drug adduct transport by ABC transporters (*vide supra*), it is interesting that DSF reduces GSH levels [54] at least in part through the formation of complexes with its different derivatives [312].

DSF [313] affects the redox balance of the cell, to GSH oxidation [314], reducing GSH levels [54] at least in part through the formation of complexes with its different

#### **Figure 3.**

*Putative mechanisms of action of disulfiram (DSF) or diethyldithiocarbamate (DETC) in combination with trypanocides in T. cruzi infection. Benznidazole (BZ) and nifurtimox are toxic and produce adverse reactions (***1***), which are ameliorated via detoxification mediated by DSF or DETC (***2***). The anti-T. cruzi agents trigger the formation of reactive oxygen species (ROS,* **3***) via nitroanion radicals (RNO2* •*− ) that give rise to superoxide (O2* •*− ), that is detoxified by superoxide dismutase (SOD,* **5***), generating hydrogen peroxide (H2O2), which in the presence of iron can produce hydroxyl radicals (*• *OH) and hydroxide anions (<sup>−</sup> OH) via Fenton reaction. DETC inhibits SOD (***4***). ROS may be detoxified by reaction with sulfhydryl or thiol groups of trypanothione (N1,N8 bis(glutathionyl)spermidine,* **6***), and this adduct can be removed by reaction with thiols of DSF/DETC (***7***). The BZ molecules in the parasite cytoplasm are extruded from the cell via p-glycoproteins or MDR transporters (***8***), which are inhibited by DETC (***9***), presumably reversing resistance phenotypes.*

derivatives [312, 315]. DETC can also reduce the GSH/non-protein thiol levels, also leading to the reduction of glutathione peroxidase activities [53, 316].

The combinations tested here may also contribute to resistance reversal, also through DETC-mediated inhibition of Fe-dependent SOD, which is linked to resistance to BZ in *T. cruzi* [66, 162, 163].

Furthermore, DSF can be used against cancer cells targeting the ubiquitin-proteasome system [317], and the ubiquitin-proteasome pathway is a therapeutic target in *T. cruzi* [318].

In this way, the strategy based of combinations of the repositioned drugs proposed here can achieve effectiveness, with selectivity and, therefore, safety in the CD treatment and sheds new light on perspectives for new therapeutic strategies.
