Panayiotis V. Ioannou

Preparation and properties of arsonolipid containing liposomes

Arsonolipids are analogs of phosphonolipids which have a chemically versatile head group. In preliminary cell culture studies, liposomes composed solely of arsonolipids or of phosholipid-arsonolipid mixtures, demonstrate a specific toxicity against cancer cells (Gortzi et al., unpublished results). The possibility of using such formulations as an alternative of arsenic trioxide with or without combination of other cytostatic agents (encapsulated in their aqueous interior) prompted the investigation of their physicochemical characteristics. Herein we compared the characteristics of arsonolipid containing vesicles with different lipid compositions. Experimental results and morphological observations reveal that non-sonicated formulations have different structures and stability (when both membrane integrity and aggregation are taken into account) depending on the acyl chain length of the arsonolipid. When phospholipids and especially cholesterol are included in their membranes almost all arsonolipids studied produce more stable vesicles. An interesting aspect of these arsonolipid containing vesicles is also their negative surface charge, which may be modulated by mixing phospholipids with arsonolipids. Sonicated vesicles have smaller sizes and profoundly higher stability, especially when containing cholesterol and phosphatidylcholine mixed with arsonolipids. The only exception is that of the arsonolipid with the C(12) acyl chain which was observed to produce long tubes which break down to cubes by sonication. In conclusion, these initial studies demonstrate that sonicated vesicles composed of arsonolipid and phospholipid mixtures mixed with cholesterol posses the stability required to be used as an arsonolipid delivery system. In addition, although cryo-electron microscopy demonstrated that the sonicated vesicles are elliptical in shape, their encapsulation efficiency is not significantly lower than sonicated phospholipid liposomes. Thereby, these vesicles may be also used for the delivery of other drug molecules which can be sufficiently retained in their aqueous interior.

REACTION OF ARSENIC(III) OXIDE, ARSENOUS AND ARSENIC ACIDS WITH THIOLS

Abstract Arsenic(III) oxide and arsenous acid in water or aqueous ethanolic solutions react, at room temperature, with a variety of lipophilic and hydrophilic thiols giving quantitatively triaryl and trialkyl trithioarsenites, (ArS)3As and (RS)3As. Aqueous solutions of arsenic acid react with certain thiols giving quantitatively mixtures of trithioarsenites and disulfides, RSSR. These reactions may help towards the elucidation of the biochemistry of arsenous and arsenic acids.

ONE POT SYNTHESIS OF ARSONOLIPIDS VIA THIOARSENITE PRECURSORS

Abstract Reduction of the racemic and optically active 1,2-dihydroxypropyl-3-arsonic acids by thiophenol gives the chloroform soluble phenyl esters of rac-, (R-, and (S)-1,2-dihydroxypropyl thioarsonous acids which are acylated by fatty acid chlorides/pyridine to give the racemic and optically active arsonolipids (1,2-diacyloxypropyl-3-arsonic acids). The yields of long chain arsonolipids are significantly higher than those reported from the acylation of salts of 1,2-dihydroxypropyl-3-arsonic acid. The yield of short chain arsonolipids are lower, partly because of difficulties in removing traces of coprecipitated fatty acids. The esters of alkylthioarsonous acids are decomposed by carboxylic acids and on silica gel and a mechanism for their acylation is proposed. The melting points of arsonolipids [(R) = (S) > rac] increase smoothly with the fatty chain length while the enthalpies of fussion [(R) > (S) > rac] show a maximum at 1,2-dimyristoyloxypropyl-3-arsonic acid. The thermal behaviour of the aqueous d...

Arsonoliposomes, a novel class of arsenic-containing liposomes: Effect of palmitoyl-arsonolipid-containing liposomes on the viability of cancer and normal cells in culture

AbstractPurpose. Arsonolipid-containing liposomes have been recently prepared. The demonstrated antileukemic action of arsenic trioxide prompted us to study their effect on the viability of several types of cancer cells to investigate the possibility of relevant applications. Five different cell types, three malignant (HL-60, C6, and GH3) and two non-malignant (HUVEC and RAME), were used. Methods. Liposomes containing the palmitoyl side chain arsonolipid (with different lipid compositions) were incubated with a given number of cells. Cell viability was estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide assay. Morphologic studies were also performed. Results. Our results reveal that arsonoliposomes cause a dose (initiated at 10−6 M)- and time-dependent inhibition of survival in all three malignant cell lines studied. No significant effect on the survival of the normal cells studied was observed at these, as well as at 10-fold higher, concentrations, although arsenic trioxide was toxic to HUVEC cells at equivalent arsenic concentrations. Microscopy studies reveal that although morphologic changes were initiated in HL-60 and C6 cells after incubation with arsonoliposomes, no changes in HUVEC and RAME cells were observed. Conclusions. Considering the numerous advantages of liposomal systems in therapeutics, it is concluded that the arsonoliposome system is very interesting and future applications should be exploited by further studies.

1,2-DIHYDROXYPROPYL-3-ARSONIC ACID: A KEY INTERMEDIATE FOR ARSONOLIPIDS

Abstract The synthesis and properties of a key intermediate for arsonolipids, rac-1,2-dihydroxypropyl-3-arsonic acid and its salts, are described. The mechanism of the Meyer reaction with β-hydroxy alkyl halides is discussed.

SYNTHESIS OF (R)- AND (S)-1,2-DIACYLOXYPROPYL-3-ARSONIC ACIDS: OPTICALLY ACTIVE ARSONOLIPIDS

Abstract Reaction of trisodium arsenite with (R)- and (S)-glycidol affords in good yields the (R)- and (S)-1,2-dihydroxypropyl-3-arsonic acid, the tetrabutylammonium salt of which upon acylation with myristic, palmitic and stearic anhydrides in the presence of pyridine gives, in moderate to low yields, optically active arsonolipids, i.e., (R)- and (S)-1,2-diacyloxypropyl-3-arsonic acids. The thermotropic phase transitions of these arsonolipids are characterized. Results show that the transition temperatures and enthalpies of fusion of the racemates are lower than those of the enantiomers in the powder form presumably because of conglomerate formation. The thermotropic transition characteristics of the aqueous dispersions of the arsonolipids change with the acyl chain lengths, and the overall behaviour is similar to that of the aqueous dispersions of phospholipids.

Arsonoliposomes: effect of arsonolipid acyl chain length and vesicle composition on their toxicity towards cancer and normal cells in culture

Arsonolipid-containing liposomes were investigated in order to characterize the influence of the lipid acyl-chain length and liposome composition on cytotoxicity. Three types of cancer cells (HL-60, C6 and GH3), and two types of normal cells (HUVEC and RAME) were used. Liposomes containing the lauroyl, myristoyl and stearoyl side chain arsonolipids (with different lipid compositions) were incubated with a given number of cells and cell viability was estimated (MTT assay and trypan blue exclusion). Morphological studies were also performed in some cases. In addition, the interaction between some of the prepared arsonoliposomes and HUVEC cells was assessed. Results reveal that all the studied arsonoliposomes cause a dose dependent inhibition of survival in all three malignant cell lines studied (initiated at 10(-6) M). The corresponding toxicity against normal cells (HUVEC and RAME) is much lower for all arsonoliposomes, except for the lauroyl side chain arsonoliposomes which were demonstrated to be relatively toxic towards normal cells, especially RAME. The microscopic observations that these vesicles possibly cause apoptosis of most cell types studied, as well as the different speed of their cytotoxic activity, imply a different mechanism of action for this arsonoliposome type. Taking the results of this study in conjunction with our previous results on arsonoliposome physical stability and cytotoxicity, it is recommended that palmitoyl-arsonolipid arsonoliposomes be used for further investigations in vivo towards the development of an anticancer product.

rac-1,2-diacyloxypropyl-3-arsonic acids arsonolipid analogues of phosphonolipids

Abstract A new class of lipids, arsonolipids or rac-1,2-diacyloxypropyl-3-arsonic acids, have been synthesized by acylating tetrabutyl ammonium hydrogen rac-1,2-dihydroxypropyl-3-arsonate with myristic, palmitic and stearic anhydrides in the presence of pyridine. The yields are moderate to low due to decomposition of rac-1,2-dihydroxypropyl-3-arsonic acid and its salts by acylating agents. Acid salts of arsonolipids were prepared by neutralization but neutral salts could not be made.

In-vitro antileishmanial and trypanocidal activities of arsonoliposomes and preliminary in-vivo distribution in BALB/c mice

We have studied the antiprotozoal activity of some recently prepared and characterized arsonoliposome formulations. Plain arsonoliposomes and phosphatidylcholine arsonoliposomes prepared with palmitoyl‐ (C16) or lauroyl‐(C12) acyl side chain arsonolipids showed in‐vitro antileishmanial activity after a 72‐h incubation period against wild‐type promastigote forms of Leishmania donovani. The IC50 values ranged from 0.40 to 11.6 μm arsonolipid. Interestingly, all preparations tested were found to be significantly more potent against amphotericin B‐ or miltefosine‐resistant promastigote forms of L. donovani, with IC50 values ranging between 0.21‐ and 2.33‐μm arsonolipid. When tested in‐vitro against Trypanosoma brucei brucei, all arsonoliposome formulations were found to have anti‐trypanosomal activity after a 24‐h incubation period. The fact that the corresponding arsonolipids (dissolved in dimethyl sulfoxide) were found not to be potent against the Leishmania promastigotes or the trypanosomes tested suggested that the formation of liposomes possibly influenced the mode of interaction between the active lipid and the parasites modulating their potency. In addition, a preliminary in‐vivo study in BALB/c mice was performed for the initial evaluation of the biodistribution of arsonoliposomes. The accumulation of arsenic in the BALB/c mouse liver in relatively high amounts was an additional advantage of this approach for anti‐protozoal therapy, especially for visceral leishmaniasis where parasites are located mainly in the liver.

Selenium status of cancer patients in Greece

The Se status of 177 Greek cancer patients has been assessed by fluorimetric analysis of the Se concentration in whole blood, urine (morning) and hair. Patients either with newly diagnosed cancers or with metastases or who are undergoing therapy have statistically significant less blood, urine and hair Se than age- and sex-matched healthy controls. There is a strong and significant correlation between blood and urine or hair Se in the newly diagnosed cancers. In cancers of systems involving absorption, metabolism and excretion of Se, and in female breast cancer, the Se levels in blood, urine and hair are significantly lower than in the controls. In cancers of the respiratory and haematologic systems, blood and urine Se are normal, while hair Se is significantly lower than in controls. The results are compared with literature data; conflicting data may be due to different dietary habits of the various populations. Some cancerous tissues, such as those of the peptic system, accumulate Se compared with their adjacent, apparently healthy tissues, but others, of the urinary system for example, do not. The reason(s) for this behaviour, however, is not known.