In Vitro Activity of Squaramides and Acyclic Polyamine Derivatives against Trophozoites and Cysts of Acanthamoeba castellanii

Pathogenic strains of Acanthamoeba cause keratitis (AK), granulomatous amoebic encephalitis (GAE), amoebic pneumonitis (AP), and skin infection in human and animals. The treatment of an Acanthamoeba infection is inva-riably very difficult and not always effective, and compounds that are amebi-cidic or amebistatic are frequently toxic and/or irritating for humans. Squaramides and polyamine derivatives have been demonstrated to have antitumor and antiprotozoal activity. The aim of this study was to investigate the activity of 5 squaramides and 5 acyclic polyamines against trophozoites and cysts of A. castellanii Neff. Amoebicidal activity against the trophozoites and cytotoxicity against Vero cells were evaluated with a colorimetric assay, using Alamar Blue®, and chlorhexidine digluconate was assayed as the reference drug. The squaramides 3 and 5 and the acyclic polyamine 6 appeared to be the most active against the trophozoites and their cytotoxicity was low, showing selectivity indexes of 28.3, 26, and 25.7, respectively, similar to the control drug, chlorhexidine digluconate (27.6). But only the squaramide 3 showed complete cysticidal activity at the concentrations of 100 and 200 µM, as the chlorhexidine digluconate. Further studies of the mechanism of action and in vivo assays are needed, but squaramide 3 could be used for developing novel therapeutic approaches against Acanthamoeba infections.


Introduction
Acanthamoeba is a free-living opportunistic protozoan parasite that is distributed in diverse environments including air, soil, freshwater, seawater, tap water, bottled mineral water, laboratory distilled water wash bottles, chlorinated swimming pools, and sewage. In addition, Acanthamoeba is known to among the most ubiquitous organisms that can be isolated from fish, reptiles, birds, and mammals [1] [2] [3] [4]. In fact, over 80% of immunocompetent individuals bear serum antibodies against Acanthamoeba antigens [5], clearly suggesting our common exposure to this parasite. Some Acanthamoeba species, such as Acanthamoeba castellanii, can cause amoebic keratitis (AK) in immunocompetent and immunocompromised individuals [6] [7], which is often associated with contact-lens wearers, as first reported by Naginton et al. [8]. On the other hand, immunocompromised individuals, including patients with AIDS, diabetes, lupus erythematosus or chemotherapy disorders, can develop granulomatous amoebic encephalitis (GAE) [9] [10]. However, this type of encephalitis is rarely observed in healthy people [11]. Amoebic pneumonitis (AP) as well as chronic lung and skin infection can be caused by Acanthamoeba, too. In addition, Acanthamoeba can serve as reservoirs for other pathogenic bacteria that are able to divide or simply survive within the cysts, such as Pseudomonas, Legionella, Mycobacterium, and Francisella tularensis [12] [13] [14].
Today, the therapy of these diseases is still problematic due to the lack of sufficiently effective drugs, resistance of the amoebas, variable efficacy between strains or species, and toxicity.
Therefore, there is a clear need for new anti-Acanthamoeba drugs. In recent years, many new treatments, natural and synthetic, have been tested with different degrees of success [15]- [24].
In this study, we have evaluated the effectiveness of several squaramides and acyclic polyamine compounds of new synthesis against trophozoites and cysts of Acanthamoeba castellanii Neff strain. Squaramide-based compounds have been demonstrated to be therapeutic, and we have described some series of oligomeric cyclosquaramides as kinase inhibitors with antitumor activity [25]. The antiparasitic activity of squaramides has been demonstrated, also, some showing antimalarial [26] and others antichagasic activity [27]. On the other hand, several macrocyclic scorpiand-like polyamines have proved effective against both the acute and chronic phases of Chagas disease [28]. Cyst forms were obtained as previously described, [30]. Briefly, Acanthamoeba trophozoites medium CGV was removed and 8% glucose in Roswell Park Memorial Institute medium (RPMI) 1640 was added. Amoebas were incubated at 30˚C for up to 48 h, followed by the addition of Sodium Dodecyl Sulfate (SDS) (0.5% final conc.). The trophozoites are SDS-sensitive and are immediately lysed upon addition of SDS, while cysts are SDS-resistant and remain intact [30].

Drugs Tested
The compounds tested were the squaramides and acyclic polyamines shown in Table 1. Squaramides 1 to 5 have a common side chain consisting of a N-(3- Chlorhexidine digluconate (Sigma, Aldrich Ltd) was used as the control drug against A. castellanii and was prepared in the same way as the squaramides and acyclic polyamines.
The solutions of the compounds were stored at −20˚C [27] [28]. These squaramides and acyclic polyamines are stable in this medium and can be stored without decomposition for long periods of time.

In Vitro Amoebicidal Assays
AlamarBlue Assay Reagent® was used in order to determine the A. castellanii anti-trophozoite activity of the squaramides and acyclic polyamines as previously described [15] [31] with some modifications.Assays were conducted using a 96-well microtiter plate (Sigma, Aldrich). Briefly, 1 × 10 4 trophozoites, in 100 µl of CGV medium with 10% inactivated fetal bovine serum, were seeded in the microtiter plates and were allowed to adhere for incubation 1 h at 28˚C.  Table 1. Squaramides and Acyclic Polyamines tested against Acanthamoeba castellanii.

Chemical structure
Chemical name were subsequently analyzed using a test wavelength of 570 nm and a reference wavelength of 630 nm with a microplate absorbance reader (Sunrise TM , Tecan).
Chlorhexidine digluconate (Sigma, Aldrich Ltd) was used as reference drug

Cysticidal Activity Assays
Acanthamoeba castellanii cysts were obtained according to Cordingley et al. [30]. Controls only with cysts, without drugs, were tested in the same way.
All the experiments were made in quadruplicate

Cytotoxicity Test on Vero Cells
The cytotoxicity induced by each compound was tested with Vero cells (ATCC

Results and Discussion
Amoebic keratitis (AK) is normally treated with a combination of a diamidine  [20].
For a better treatment against Acanthamoeba, we assayed two groups of compounds: squaramides and acyclic polyamines (Table 1). Antiparasitic activity (antimalarial and anti-Chagas) has been demonstrated for some of them [26] [27] [28]. Some aminosquaramide compounds induced changes in the T. cruzi glycolysis cycle and caused cytoplasmic and mitochondrial alterations [27] [28].
The cytotoxic activity of the compounds was assayed against mammalian Vero cells and the selective index (IC 50 Vero cells toxicity/IC 50 activity on trophozoites forms of the parasite) was calculated for each drugs.
AlamarBlue Assay Reagent® was used to determine the anti-trophozoite, of A. castellanii, and cytotoxic activity. This technique was used because it is easy, sensitive and cheap. Resazurin, which is the active ingredient of AlamarBlue® reagent, is a non-toxic, cell-permeable compound that is blue colored and virtually non-fluorescent. Upon entering cells, resazurin is reduced to resorufin, a compound that is red in color and highly fluorescent. Viable cells continuously convert resazurin to resorufin, intensifying the overall fluorescence and color of the media surrounding cells.
The in vitro trophocidal activity, cytotoxicity, and selectivity indexes are summarised in Table 2. The analysis of these results reveals that two squaramides, 3 and 5, and two acyclic polyamines, 6 and 7, were the most effective against trophozoites of A. castellanii. Squaramide 3 gave the lowest IC 50 value, 3.5 ± 0.5 µM vs. 5.3 ± 3.1 µM of the chlorhexidine digluconate, and its selectivity index was higher than for the control drug (28.3 vs. 27.64). Squaramide 5 with IC 50 of 11.4 ± 1.2 and selectivity index of 26 showed lower trophocidal activity than that of squaramide 3 and the control drug but was less cytotoxic. Both squaramides feature similar structures but different substitution patterns of the cyclobutendione moiety. Squaramide 3 is a N, N-bisubstituted compound while squaramide 5 is a N,O squaramide (sometimes referred as squaramide-esters). Thus, the substitution of a NH group by O markedly disturbs the trophocidal activity but, at the same time, drastically reduces the cytotoxicity of 5. It is worth noting the parallelism between squaramide 3 and 5 and miltefosine, MF, ( Figure  1). Miltefosine is a medication used mainly to treat leishmaniasis [37] and free-living amoeba infections such as granulomatous amoebic encephalitis caused by Acanthamoeba spp. and Balamuthia mandrillaris and primary amoebic meningoencephalitis caused by Naegleria fowleri [38] [39].   after treatment with miltefosine [39] [40]. In 2016, after treatment that included miltefosine, another child became the fourth person in the United States to survive Naegleria fowleri infection [41].
In the neutral or slightly acidic intracellular cytoplasmatic environment of the parasite, squaramide 3 and 5 are heavily protonated at the dimethyl amino group. On the other hand, the squaramide moiety is known to act as a successful masked phosphate group in bioisosteric replacement [42] [43].
Although all the acyclic polyamines (6-10) were less effective against the trophozoites than the squaramides, their cytotoxicity against Vero cells was very  Table 1).
The effect of the drugs against the trophozoites was observed by inverted microscopy. Figure 2 shows the appearance of the trophozoites at the lowest concentration tested, 5 µM, after 120 h of treatment with chlorhexidine digluconate (Figure 2(b)) and with the most effective compounds: 3 (Figure 2(c)), 5 ( Figure   2(d)), and 6 ( Figure 2(e)). It was observed that the alterations were dose dependent, but at the lower concentration (5 µM) many trophozoites were detached and dead, appearing agglutinated and floating in the supernatant, especially with chlorhexidine digluconate (Figure 2 shows trophozoites treated with polyamine 6 with similar appearance to that treated with 5 µM concentration. After the study of the amoebicidal and cytoxic activity of the drugs, squaramides 3 and 5 and polyamine 6 were selected to be tested against A. castellanii cysts, which are less susceptible to the treatments than are trophozoites. Although in many recent publications on chemotherapy against Acanthamoeba, [15] [17] [18] [22] [24] [44], only the amoebicidal activity of the different products is assayed, it has become evident that it is necessary to know the cysticidal activity because trophozoites and cysts are present in the eyes, lungs, brain, and skin of the patients, and a good treatment against the trophozoite is not always effective against cysts, which are more resistant. Figure 3 shows the cysticidal activity of the chlorhexidine digluconate (Figure 3(a)) and compounds 3, 5 and 6 ( Figures 3(b)-(d), respectively) versus a control of cysts without treatment.
The results showed that no cysts reverted to trophozoites with chlorhexidine digluconate and compound 3, (Figure 3(a) and Figure 3(b)), at 100 and 200 µM during of 192 h that the study lasted, and therefore they were presumed to be non-viable. When cysts were treated with 50 µM of these drugs, very few trophozoites (no more than 9%) could be seen. The cysts treated with compound 5 and 6 reverted with time to trophozoites at the concentrations assayed ( Figure   3(a) and Figure 3(d)).
Chlorhexidine digluconate is used at 0.02% concentration in the initial thera-   amoebas, with irreversible loss of calcium and cell electrolytes from the cytoplasm, causing cell lysis and death [32]. However, it is difficult to believe that a chemical destroying the membrane of the amoeba would not at the same time affect the plasma membranes of the ocular cells, and it has been demonstrated that with continued medical treatment, the iris and lens cells die, and cataracts develop [45]. Also, the cysticidal activity of chlorhexidine digluconate at 0.02% is only of the 80% [44]. In this study, cysticidal activity of chlorhexidine digluconate and squaramide 3 was similar, but squaramide 3 was more effective against the trophozoites and thus served as an alternative in the treatment of Acantha-moeba infections, (AK, GAE, EP), according to in vivo tests. However, chlorhexidine digluconate has shown poor corneal penetration into the anterior chamber after topical administration [46].
Squaramides and acyclic polyamines were selected, placing emphasis on the molecular properties that determine drugability but also on the commercial availability and affordability of the starting reagents and the production process.
These factors are coupled with a short synthesis and simple purification steps as well as low cost.
In conclusion, some of the squaramides and acyclic polyamines tested exert effective amoebicidal and/or cysticidal activity against A. castellanii Neff in vitro, comparable to control drug with less cytotoxicity, especially squaramide 3 with