Martin Mrva

Synthesis and biological activity of dialkylphosphocholines

A series of dialkylphosphocholines were prepared and evaluated for their biological activity. The antiprotozoal activity was determined against Acanthamoeba lugdunensis. Compound 15 exhibited excellent trophocidal activity. None of the tested dialkylphosphocholines exhibited better fungicidal activity against Candida albicans than miltefosine. The antineoplastic activity was determined against HeLa. The most cytotoxic was compound 10, which was more active against tumor cells as against normal cells.

Synthesis, self-aggregation and biological properties of alkylphosphocholine and alkylphosphohomocholine derivatives of cetyltrimethylammonium bromide, cetylpyridinium bromide, benzalkonium bromide (C16) and benzethonium chloride

A series of alkylphosphocholine and alkylphosphohomocholine derivatives of cetyltrimethylammonium bromide, cetylpyridinium bromide, benzalkonium bromide (C16) and benzethonium chloride have been synthesized. Their physicochemical properties were also investigated. The critical micelle concentration (cmc), the surface tension value at the cmc (γcmc), and the surface area at the surface saturation per head group (Acmc) were determined by means of surface tension measurements. The prepared compounds exhibit significant cytotoxic, antifungal and antiprotozoal activities. Alkylphosphocholines and alkylphosphohomocholines possess higher antifungal activity against Candida albicans in comparison with quaternary ammonium compounds in general. However, quaternary ammonium compounds exhibit significantly higher activity against human tumor cells and pathogenic free-living amoebae Acanthamoeba lugdunensis and Acanthamoeba quina compared to alkylphosphocholines. The relationship between structure, physicochemical properties and biological activity of the tested compounds is discussed.

Weak Cytotoxic Activity of Miltefosine Against Clinical Isolates of Acanthamoeba spp.

Abstract Hexadecylphosphocholine (miltefosine) is an anticancer drug active in vitro against various protozoan parasites, and recently used for the treatment of disseminated Acanthamoeba infection. In the present study, we present results of weak cytotoxic activity of this potential amoebicidal agent for 2 of 3 clinical isolates of Acanthamoeba spp. Although the inhibition effect for all tested concentrations was apparent, and showed 100% eradication of trophozoites of Acanthamoeba castellanii strain at a concentration of 62.5 µM after 24 hr, the strains Acanthamoeba sp. and Acanthamoeba lugdunensis exhibited low sensitivity to hexadecylphosphocholine, even in high concentrations. The determined minimal trophocidal concentrations were 250 µM for Acanthamoeba sp. and 500 µM for A. lugdunensis after 24 hr of exposure. Although hexadecylphosphocholine is a potential agent for treatment of Acanthamoeba keratitis and systemic infections, in clinical practice the possible insusceptibility of the amoebic strain should be considered for optimizing therapy.

Relationship between aggregation properties and antimicrobial activities of alkylphosphocholines with branched alkyl chains

Synthesis of five alkylphosphocholines with branched alkyl chains (Isophol-PCs) with different length of alkyl chains was described. Isophol(8)-PC and Isophol(12)-PC represent new compounds. The physico-chemical properties of Isophol-PCs were determined, critical micelle concentration and types of formed aggregates in aqueous solutions were investigated. The biological activities of Isophol-PCs have been studied for the first time in the present study. Antimicrobial activities of alkylphosphocholines were studied against bacteria (Staphylococcus aureus, Escherichia coli), yeast (Candida albicans) and pathogenic free-living amoebae (Acanthamoeba lugdunensis and Acanthamoeba quina). A. lugdunensis and A. quina are relatively insusceptible to action of miltefosine (standard compound of alkylphosphocholines) and therefore they are good models for studies of amoebicidal action of the investigated compounds. Relationship between structure, physico-chemical and biological activities of Isophol-PCs was discussed. S. aureus and C. albicans were sensitive to action of Isophol(16)-PC, Isophol(20)-PC. E. coli was not sensitive to action of all studied alkylphosphocholines in the concentrations equal to, or less than 10mM. Among all the synthesized compounds, Isophol(16)-PC had the highest level of activity against both strains of Acanthamoeba. The minimum trophocidal concentrations of Isophol(16)-PC against A. lugdunensis and A. quina are about four times lower than the minimum trophocidal concentrations of miltefosine against both strains.

Phylogeny and Systematics of Leptomyxid Amoebae (Amoebozoa, Tubulinea, Leptomyxida)

We describe four new species of Flabellula, Leptomyxa and Rhizamoeba and publish new SSU rRNA gene and actin gene sequences of leptomyxids. Using these data we provide the most comprehensive SSU phylogeny of leptomyxids to date. Based on the analyses of morphological data and results of the SSU rRNA gene phylogeny we suggest changes in the systematics of the order Leptomyxida (Amoebozoa: Lobosa: Tubulinea). We propose to merge the genera Flabellula and Paraflabellula (the genus Flabellula remains valid by priority rule). The genus Rhizamoeba is evidently polyphyletic in all phylogenetic trees; we suggest retaining the generic name Rhizamoeba for the group unifying R. saxonica, R.matisi n. sp. and R. polyura, the latter remains the type species of the genus Rhizamoeba. Based on molecular and morphological evidence we move all remaining Rhizamoeba species to the genus Leptomyxa. New family Rhizamoebidae is established here in order to avoid paraphyly of the family Leptomyxidae. With the suggested changes both molecular and morphological systems of the order Leptomyxida are now fully congruent to each other.

Synthesis of structural analogues of hexadecylphosphocholine and their antineoplastic, antimicrobial and amoebicidal activity

Twelve derivatives of hexadecylphosphocholine (miltefosine) were synthesized to determine how the position and length of the alkyl chain within the molecule influence their biological activities. The prepared alkylphosphocholines have the same molecular formula as miltefosine. Activity of the compounds was studied against a spectrum of tumour cells, two species of protozoans, bacteria and yeast. Antitumour efficacy of some alkylphosphocholines measured up on MCF-7, A2780, HUT-78 and THP-1 cell lines was higher than that of miltefosine. The compounds showed antiprotozoal activity against Acanthamoeba lugdunensis and Acanthamoeba quina. Some of them also possess fungicidal activity against Candida albicans equal to miltefosine. No antibacterial activity was observed against Staphylococcus aureus and Escherichia coli. A difference in position of a long hydrocarbon chain within the structure with maximum efficacy was observed for antitumour, antiprotozoal and antifungal activity.

Mayorella vespertilioides Page, 1983 (Amoebozoa) — new host for the ectoparasitic fungus Amoebophilus simplex (Zygomycota)

During research on gymnamoebae in freshwater ponds near the Modra town (Slovakia), in one sample of water and sediment of the 71 samples examined a fungal infection of Amoebophilus simplex Barron, 1983 (Zygomycota: Zoopagales) in Mayorella vespertilioides Page, 1983 (Amoebozoa: Flabellinea) was detected for the first time. From 27 observed amoebae, 9 specimens (33%) were infected. Populations of other species recorded in the sample, Mayorella penardi and Korotnevella stella, were uninfected.

Observations on morphology of insufficiently known amoebae Saccamoeba wellneri Siemensma, 1987 and Saccamoeba limna Bovee, 1972 (Gymnamoebia: Hartmannellidae)

The morphology of poorly known freshwater amoebae, Saccamoeba wellneri Siemensma, 1987 and Saccamoeba limna Bovee, 1972, were investigated using living observations at light microscopic level. S. wellneri was collected from Karloveské rameno branch of the Danube River and Štrkovecké jazero lake in Bratislava, Slovakia. Its locomotive form is typical with crescent-shaped and bipyramidal crystals and uroid with short wart-like papillae. S. limna was found in an indoor freshwater aquarium in Bratislava, Slovakia. The locomotive form of S. limna has broad monopodial shape and possesses typical hyaline uroid with short nonadhesive filaments. Many small bipyramidal crystals were noted. Additional data and details on the morphology of both species were described and illustrated and re-diagnoses are presented. This paper is helpful for their identification on the base of locomotive morphology, floating, and resting forms and allows distinguishing S. wellneri and S. limna from other hartmannellids at light microscopic level.

Cytomorphological changes and susceptibility of clinical isolates of Acanthamoeba spp. to heterocyclic alkylphosphocholines.

The treatment of diseases caused by pathogenic strains of Acanthamoeba spp. is to date limited and frequently unsuccessful. Alkylphosphocholines (APCs) are promising agents with interesting results of antiparasitic activity in experimental and clinical conditions. In the present study susceptibilities of two clinical isolates of Acanthamoeba spp. to four heterocyclic APCs were investigated. The isolates showed high degrees of susceptibility to studied APCs and all the tested concentrations inhibited the growth with the highest concentrations of 500-1000μM causing 100% eradication of the trophozoites and cysts. The highest susceptibility was noted in IF16-P-4-Pip with EC50 values of 28.62-43.73μM, and EC90 values of 30.70-63.16μM after 48h of incubation. The cytomorphological changes of trophozoites after the exposure to APCs included rounding up of cells, resorption of acanthopodia and subsequent lysis. The remains of cells were typical with oval shape and identifiable nucleus. After the application of IF16-P-4-Pip, IF16-P-2-MetPip, and IF16-P-Azep, at concentrations of 62.5-125μM to trophozoite suspension, a formation of pseudocysts was detected. The single-layered coat covering the surface of pseudocyst stained positively with a fluorescence brightener, Rylux. Destroyed cysts were characteristic with shrinkage of the cytoplasm and separation of the cytoplasmic membrane from the endocyst. IF16-P-2-MetPip at the highest concentration formed large spherical vesicles which frequently enclosed inactivated cysts. Heterocyclic APCs used in the study demonstrated strong amoebicidal activity and the cytotoxic effect of IF16-P-4-Pip similar to that of miltefosine indicates its possible therapeutic potential.

Diversity of gymnamoebae (Rhizopoda, Gymnamoebia) in a rain-water pool

The diversity of species of gymnamoebae in a single sample from a rain-water pool in Bratislava (Slovakia) was investigated. To maximize the recovery of species, six enrichment cultivation media were used. A total of 12 taxa, representing five families and eight genera of Gymnamoebia, were recovered.