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 . In fact, over 80% of immunocompetent individuals bear serum antibodies against Acanthamoeba antigens , 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 , which is often associated with contact-lens wearers, as first reported by Naginton et al. . On the other hand, immunocompromised individuals, including patients with AIDS, diabetes, lupus erythematosus or chemotherapy disorders, can develop granulomatous amoebic encephalitis (GAE) . However, this type of encephalitis is rarely observed in healthy people . 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 .
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 - .
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 . The antiparasitic activity of squaramides has been demonstrated, also, some showing antimalarial and others antichagasic activity . On the other hand, several macrocyclic scorpiand-like polyamines have proved effective against both the acute and chronic phases of Chagas disease .
2. Materials and Methods
2.1. Culture of Acanthamoeba castellanii Trophozoites and Encystment
Acanthamoeba castellanii Neff (ATCC 30010), obtained from The Center of Scientific Instrumentation of the University of Granada, was used in this study.
Trophozoites were axenically cultured in Falcon Flasks with Casitone Glucose Vitamins medium (CGV) supplemented with 10% inactivated fetal calf serum, , at 28˚C.
Cyst forms were obtained as previously described, . 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 .
2.2. 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-(dimetylaminopropyl)-N-methyl residue. This residue has proven critical for the design of squaramide-based antichagasic agents . The second arm of the squaramide consisted of aliphatic chains with increasing lengths aimed at improving the lipophilicity of the squaramides. The amines tested are ethylenediamine derivatives with amine group mono- or dimethylated amine and the other one functionalized with a methyl pyridine substituent.
For amoebicidal assays, squaramides and acyclic polyamines were prepared at different concentrations of 400, 200, 100, 50, 25, and 10 µM in CGV medium with 10% inactivated fetal bovine serum. The drugs were prepared at 200, 100, 50, and 10 µM concentrations in RPMI medium with 8% glucose for the studies of cysticidal activity. For cytotoxicity test on Vero cells the drugs were prepared at 800, 400, 200, 100, and 50 µM concentrations in RPMI with 10% inactivated fetal bovine serum.
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 . These squaramides and acyclic polyamines are stable in this medium and can be stored without decomposition for long periods of time.
2.3. 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 with some modifications.Assays were conducted using a 96-well microtiter plate (Sigma, Aldrich). Briefly, 1 × 104 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.
After that, 100 µl of the solutions of the squaramides or acyclic polyamines prepared at 400, 200, 100, 50, 25, and 10 µM were added to each well. AlamarBlue Assay Reagent® (Thermo Fisher Scientific) was placed into each well at 10% of the final volume, 20 µl. Test plates were incubated for 120 h at 28˚C in complete darkness to avoid the oxidation of the resazurin with the light. The plates
Table 1. Squaramides and Acyclic Polyamines tested against Acanthamoeba castellanii.
were subsequently analyzed using a test wavelength of 570 nm and a reference wavelength of 630 nm with a microplate absorbance reader (SunriseTM, Tecan).
Chlorhexidine digluconate (Sigma, Aldrich Ltd) was used as reference drug against A. castellanii trophozoites. This control drugs and other controls only with trophozoites, without drugs, were tested in the same way.
All the experiments were performed in quadruplicate and the concentration required to give 50% inhibition (IC50) was calculated by linear regression analysis from Kc values at the concentrations used, (5, 12.5, 25, 50, 100, and 200 µM). Selective indexes, (IC50 Vero cells toxicity/IC50 activity on trophozoites forms of the parasite), were also calculated.
2.4. Cysticidal Activity Assays
Acanthamoeba castellanii cysts were obtained according to Cordingley et al. . Cysts were washed twice with RPMI medium. Afterwards, 1 × 104 cyst were added to each well of a microtiter plate in 200 µl of the RPMI with 8% glucose and the compounds at 200, 100, 50, and 10 µM. In the same way was tested the control drug, chlorhexidine digluconate.
The microtiter plate was incubated for 120 h at 28˚C. Finally, cysts were slightly washed and fresh CGV medium with 10% inactivated fetal bovine serum was added to each well, the microtiter plate was incubated at 28˚C, and the number of cysts and trophozoites were counted using a Neubauer chamber at different times of 24, 48, 72, 120, and 192 h. The presence of trophozoites was considered as indication of cyst viability.
Controls only with cysts, without drugs, were tested in the same way.
All the experiments were made in quadruplicate
2.5. Cytotoxicity Test on Vero Cells
The cytotoxicity induced by each compound was tested with Vero cells (ATCC CCL-81) growing in RPMI (Gibco) supplemented with 10% inactivated fetal bovine serum in a humidified 95% air and 5% CO2 atmosphere at 37˚C. Vero cells, 104 in 100 µl of medium, were added to each well of a microtiter plate and incubated for 24 h at 37˚C in a humid atmosphere enriched with 5% CO2. Next, 100 µl of the drug prepared at 800, 400, 200, 100, and 50 µM were added to each well. AlamarBlue Assay Reagent® (Thermo Fisher Scientific) was placed into each well at 10% of the final volume, 20 µl. Test plates were incubated for 120 h at 37˚C and were subsequently analyzed using a test wavelength of 570 nm and a reference wavelength of 630 nm with a microplate absorbance reader (SunriseTM, Tecan).
Vero cells were treated with chlorhexidine digluconate in the same way and a control with only Vero cells without treatment were done.
All the experiments were conducted in quadruplicate and the concentration required to give 50% inhibition (IC50) was calculated by linear regression analysis from Kc values at the concentrations used (400, 200, 100, 50 and 25 µM).
3. Results and Discussion
Amoebic keratitis (AK) is normally treated with a combination of a diamidine (hexamidine or propamidine isothionate) and a biguanide (chlorhexidine or polyhexamethylene biguanide) . However, drug resistance against AK treatment has been demonstrated and at present, there is no completely effective treatment for granulomatous amoebic encephalitis GAE .
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 . Some aminosquaramide compounds induced changes in the T. cruzi glycolysis cycle and caused cytoplasmic and mitochondrial alterations .
We assayed the amoebicidal and cysticidal activity of 5 squaramides and 5 acyclic polyamines against A. castellanii Neff vs. the reference drug, chlorhexidine digluconate, a standard antiseptic used treating Acanthamoeba keratitis with demonstrated cysticidal activity .
The cytotoxic activity of the compounds was assayed against mammalian Vero cells and the selective index (IC50 Vero cells toxicity/IC50 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 IC50 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 IC50 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 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 . In 2013, two children survived and recovered from primary amoebic meningoencephalitis
Table 2. In vitro activity, toxicity and selectivity index for squaramides and acyclic polyamines derivatives on trophozoites of Acanthamoeba castellanii.
Figure 1. Molecular structures of squaramides 3 and 5 compared to that of the miltefosine (MF).
after treatment with miltefosine . In 2016, after treatment that included miltefosine, another child became the fourth person in the United States to survive Naegleria fowleri infection .
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 .
Although all the acyclic polyamines (6-10) were less effective against the trophozoites than the squaramides, their cytotoxicity against Vero cells was very low, reaching a selectivity index of 25.7 for polyamine 6. While polyamines 6 and 7 are isomers, differing only in the disposition of the nitrogen of the pyridine ring, their differences in amoebicidal activity and cytotoxicity was remarkable, with polyamine 7 showing a selective index of only 8.87. Notably, isomeric polyamines 8-10, which differ from 6-7 in the presence of only one methyl group instead of two at one of their ends, registered much lower amoebicidal activity, (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(b)) and compound 3 (Figure 2(c)). Treated trophozoites showed structural alterations such as reduction in size, loss of acanthopodia and roundness vs. the control trophozoites (Figure 2(a)). Trophozoites treated with the highest concentration of compounds (200 µM) during 120 h are showed in Figures 2(f)-(i). They were rounded, agglutinated, without internal content, dead and many cells debris from the destruction of the trophozoites appeared floating in the supernatant Figures 2(f)-(h). Figures 2(i) 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, , 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 therapy of Acanthamoeba keratitis. It acts by damaging the membrane of the
Figure 2. a) Trophozoites control of Acanthamoeba castellanii Neff. (b)-(e) Trophozoites treated with chlorhexidine digluconate (b) and compounds 3 (c), 5 (d), 6 (e) at concentration 5 µM during 120 h. They appeared detached, rounded, agglutinated and floating in the supernatant. (f)-(i) Trophozoites treated with chorhexidine digluconate (f) and compounds 3 (g), 5 (h), 6 (i) at concentration 200 µM during 120 h. Trophozoites in (f), (g) and (h) were dead and cell debris appeared floating in the supernatant. ×400. Scale bar = 10 µm.
Figure 3. Percentage of cysts when they were treated with: (a) Chlorhexidine digluconate, (b) Squaramide 3, (c) Squaramide 5 and (d) Polyamine 6. No cysts reverted to trophozoites with chlorhexidine digluconate (a) and compound 3 (b) at 100 and 200 µM during of 192 h.
amoebas, with irreversible loss of calcium and cell electrolytes from the cytoplasm, causing cell lysis and death . 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 . Also, the cysticidal activity of chlorhexidine digluconate at 0.02% is only of the 80% . 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 Acanthamoeba infections, (AK, GAE, EP), according to in vivo tests. However, chlorhexidine digluconate has shown poor corneal penetration into the anterior chamber after topical administration .
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 IC50 of 3.5 µM against trophozoites and cysticidal activity at concentrations of 100 and 200 µM. Further chemical modifications and in vitro as well as in vivo studies are needed to understand the molecular mechanisms of action and the alterations produced in the amoebas, as well as to assess their ocular toxicity in animal models.
We would like to thank financial support from the Spanish MINECO, FEDER funds of the E. U. (Project CTQ2013-14892, CONSOLIDER INGENIO 2010-CSD2010-00065) and Unidad de Excelencia MDM (2015-0038) and Generalitat Valenciana (PROMETEO II 2015-002).
Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this paper.