Potent In Vitro Antiproliferative Synergism of Combinations of Ergosterol Biosynthesis Inhibitors against Leishmania amazonensis.

Leishmaniases comprise a spectrum of diseases caused by protozoan parasites of the Leishmania genus. Treatments available have limited safety and efficacy, high costs, and difficult administration. Thus, there is an urgent need for safer and more-effective therapies. Most trypanosomatids have an essential requirement for ergosterol and other 24-alkyl sterols, which are absent in mammalian cells. In previous studies, we showed that Leishmania amazonensis is highly susceptible to aryl-quinuclidines, such as E5700, which inhibit squalene synthase, and to the azoles itraconazole (ITZ) and posaconazole (POSA), which inhibit C-14α-demethylase. Herein, we investigated the antiproliferative, ultrastructural, and biochemical effects of combinations of E5700 with ITZ and POSA against L. amazonensis. Potent synergistic antiproliferative effects were observed against promastigotes, with fractional inhibitory concentration (FIC) ratios of 0.0525 and 0.0162 for combinations of E5700 plus ITZ and of E5700 plus POSA, respectively. Against intracellular amastigotes, FIC values were 0.175 and 0.1125 for combinations of E5700 plus ITZ and E5700 plus POSA, respectively. Marked alterations of the ultrastructure of promastigotes treated with the combinations were observed, in particular mitochondrial swelling, which was consistent with a reduction of the mitochondrial transmembrane potential, and an increase in the production of reactive oxygen species. We also observed the presence of vacuoles similar to autophagosomes in close association with mitochondria and an increase in the number of lipid bodies. Both growth arrest and ultrastructural/biochemical alterations were strictly associated with the depletion of the 14-desmethyl endogenous sterol pool. These results suggest the possibility of a novel combination therapy for the treatment of leishmaniasis.

Paracoccidioides brasiliensis, paracoccidioidomycosis, and antifungal antibiotics.

Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis (PCM), a human systemic, chronic and progressive mycosis. Preferred antifungals are sulfamethoxazol-trimethoprim, itraconazole, amphotericin B. Treatment is lengthy, the drugs may have undesirable side effects, and some are costly. Occasional resistant strains have been reported. Therefore, the search for more selective and efficient antifungals to treat this and other mycoses continues. Ajoene, chemically derived from garlic, behaves as an antifungal agent against P. brasiliensis and other fungi. Its antiproliferative effects in P. brasiliensis are associated with a reduction of phosphatidyl choline, a concomitant increase in its precursor phosphatidyl ethanolamine, and a large increase in unsaturated fatty acids in the pathogenic yeast phase. The sterol biosynthetic pathway has been largely studied for the search of antifungals. Azoles and allilamines act on differents steps of this pathway. However, they may interfere with similar steps in the host. Hence, the search for drugs that may act on more specific steps is ongoing. One such step focuses on the sterol C-methylations catalyzed by the enzyme (S)-adenosyl-L-methionine: Delta(24) - sterol methyl transferase (SMT). SMT inhibitors such as azasterols and derivatives (AZA1, AZA2, AZA3) have proven highly effective as antiproliferative agents against protozoa and some fungi, among them, P. brasiliensis. Their chemical synthesis and structure, and their molecular electrostatic potential are discussed in order to understand their mechanism of action, and derive rationally designed improvements on these molecules, that would favour a higher efficacy and selectivity.

Studies of molecular structure parameters of 20-piperidin-2-yl-5alpha-pregnan-3beta,20-diol and its N-methyl derivative: two inhibitors of Delta24(25) sterol methyl transferase and Delta24(24') sterol methyl reductase of Trypanosoma cruzi.

Molecular structural parameters of two potential drugs against Trypanosoma cruzi epimastigotes, 20-piperidin-2-yl-5alpha-pregnan-3beta,20-diol (1) and 20-N-methylpiperidin-2-yl-5alpha-pregnan-3beta, 20-diol (2) were studied using a combination of a stereoselective synthetic route, spectroscopic characterization and single-crystal X-ray analysis. Both compounds were synthesized with an R configuration at C20. This chirality is a consequence of the stereoselectivity observed during the formation of the intermediate 20-pyridin-2-yl-5alpha-pregnan-3beta,20R-diol (4). NMR data indicated that the six-membered aza ring of (2) is conformationally more restrained, in CDCl3 solution, than (1). X-ray studies showed that maximum deviations among structural molecular parameters of (1) and (2) correspond to torsion angles along the C20-C22 bonds, leading to a different relative orientation of the N atom; a critical structural parameter for the binding properties of aza-sterols to Delta(24(25)) sterol methyl transferase. Cremer-Pople parameters of the five-membered rings of (1) and (2) lie in the observed range for a family of tetracyclic fused ring systems retrieved from the CSD. The phi2 parameter of (1) lies just on the mean of the family, while phi2 of (2) deviates significantly towards the lower limit.

In vitro antiproliferative effects and mechanism of action of the new triazole derivative UR-9825 against the protozoan parasite Trypanosoma (Schizotrypanum) cruzi.

We describe the in vitro antiproliferative effects of the new triazole derivative UR-9825 against the protozoan parasite Trypanosoma (Schizotrypanum) cruzi, the causative agent of Chagas' disease in Latin America. The compound was found to be extremely active against the cultured (epimastigote) form of the parasite, equivalent to that present in the reduviid vector, with a MIC of 30 nM, a concentration 33-fold lower than that required with the reference compound ketoconazole. At that MIC, growth arrest coincided with depletion of the parasite's 4,14-desmethyl endogenous sterols (ergosterol, 24-ethylcholesta-5,7,22-trien-3b-ol, and precursors) and their replacement by methylated sterols (lanosterol, 24-methylenedihydrolanosterol, and obtusifoliol), as revealed by high-resolution gas chromatography coupled with mass spectrometry. This indicated that the primary mechanism of action of UR-9825 was inhibition of the parasite's sterol C14alpha demethylase, as seen with other azole derivatives. The phospholipid composition of growth-arrested epimastigotes was also altered, when compared to controls, with a significant increase in the content of phosphatidylethanolamine and phosphatidylserine and a concomitant reduction of the content of phosphatidylcholine. The clinically relevant intracellular amastigote form, grown in cultured Vero cells at 37 degrees C, was even more sensitive to UR-9825, with a MIC of 10 nM, comparable to that for ketoconazole. The results showed that UR-9825 is among the most potent azole derivatives tested against this parasite and support in vivo studies with this compound.

Sterol composition and biosynthesis in Trypanosoma cruzi amastigotes.

A detailed analysis of the endogenous sterols present in the clinically relevant intracellular (amastigote) stages of Trypanosoma cruzi, is presented. The parasites were grown in cultured Vero cells in the absence or presence of different sterol biosynthesis inhibitors, including the C14alpha demethylase inhibitor ketoconazole and two inhibitors of delta24(25)-sterol methyl transferase, 20 piperidin-2-yl-5alpha-pregnan-3beta-20-R-diol (22,26-azasterol) and 24-(R,S),25-epiminolanosterol. Amastigotes were isolated and purified from their host cells and neutral lipids were extracted, separated and analyzed by chromatographic and mass spectrometric methods. Control (untreated) amastigotes contained as main endogenous sterols 24-methyl-cholesta-7-en-3beta-ol (ergosta-7-en-3beta-ol) and its 24-ethyl analog, plus smaller amounts of their precursor, ergosta-7,24(28)dien-3beta-ol; these cells also contained cholesterol (up to 80% by weight of total sterols), probably derived from host cells. Amastigotes that proliferated in the presence of 10 nM ketoconazole (minimal inhibitory concentration, MIC) for 24 h had a sharply reduced content of endogenous 4-desmethyl sterols with a concomitant accumulation of 24-methyl-dihydrolanosterol and 24-methylene-dihydrolanosterol. On the other hand, amastigotes incubated during the same period of time with the two inhibitors of 24(25)-SMT at their respective MICs (100-300 nM) accumulated large amounts of C27 sterols whose structure suggested, in the case of 22,26-azasterol, that delta14 sterol reductase was also inhibited. Ketoconazole produced a dose-dependent reduction in the incorporation of [2-(14)C]-acetate into the parasite's endogenous C4-desmethyl sterols with an IC50 of 50 nM, indistinguishable from the value reported previously for the extracellular epimastigote form. Taken together, the results showed that amastigotes have a simpler sterol biosynthetic pathway than that previously described for epimastigotes, lacking both delta5 and delta22 reductases. They also suggest that the 100-fold higher potency of antifungal azoles as antiproliferative agents against amastigotes, when compared with epimastigotes, is most probably due to a smaller pool of endogenous sterols in the intracellular parasites.

Inhibitors of delta24(25) sterol methyltransferase block sterol synthesis and cell proliferation in Pneumocystis carinii.

Detailed analysis of the endogenous sterol content of purified Pneumocystis carinii preparations by gas-liquid chromatography coupled to mass spectrometry suggested that this parasite can both synthesize de novo steroid skeletons (to produce delta7 sterols) and take them from the infected host (leading to delta5 sterols). In both cases the final products are 24-alkyl sterols, resulting from the action of delta24(25) and delta24(24') sterol methyltransferases, enzymes not present in vertebrates. To investigate the physiological significance of these sterols, cultures of P. carinii in embryonic lung cells were exposed to 22,26-azasterol (20-piperidin-2-yl-5alpha-pregnan-3beta-20(R)-diol), a compound previously shown to inhibit both enzymes and to halt cell proliferation in fungi and protozoa. This compound produced a dose-dependent reduction in the parasite proliferation, with a 50% inhibitory concentration of 0.3 microM and 80% reduction of growth after 96 h at 10 microM. Correspondingly, parasites treated with the azasterol at 10 microM for 48 h accumulated 24-desalkyl sterols such as zymosterol (cholesta-8,24-dien-3beta-ol) and cholesta-8,14,24-trien-3beta-ol to ca. 40% of the total mass of endogenous sterols. This is the first report on the antiproliferative effects of a sterol biosynthesis inhibitor on P. carinii and indicate that sterol methyltransferase inhibitors could be the basis of a novel and specific chemotherapeutic approach to the treatment of P. carinii infections.

A descriptive-exploratory study of outcome and some biopsychosocial characteristics of drug dependent veterans in Puerto Rico.

OBJECTIVE: The authors attempted to correlate outcomes with various significant biopsychosocial variables in drug dependent veterans receiving outpatient treatment at San Juan V.A.M.C. METHODS: The clinical charts of 120 inactive patients were examined using a 50 item questionnaire. The data was processed and analyzed using the EpiInfo v.5 computer program. The outcome of the patients was measured using a Goal Attainment Scale Technique with demonstrated validity and reliability. RESULTS: The patients retention in treatment for 12 or more visits and its relationship to good outcomes was the most significant finding of this study (P < 0.00000000, 80.5% of patients improved). Only 5% of patients with 3 or less visits showed positive outcomes. Another variable associated with good outcomes was a supportive wife (P < 0.02). HIV reactivity was strongly correlated with IV drug use (P < 0.037). CONCLUSIONS: The strong correlation of the retention of patients in treatment for 12 or more visits and good outcomes should alert clinicians and policy makers as to the importance of available and effective treatments in the fight against substance abuse and mental health problems. This study also showed that a supportive family member is usually the best social asset that patients and therapists have in their fight for sobriety.

Modification of the sterol composition of Trypanosoma (Schizotrypanum) cruzi epimastigotes by delta 24(25)-sterol methyl transferase inhibitors and their combinations with ketoconazole.

We report a detailed analysis of the sterol composition of Trypanosoma cruzi epimastigotes grown in the absence or presence of two sterol analogs previously reported as inhibitors of delta 24(25) sterol methyltransferase (24(25)-SMT,E.C.2.1.1.43) in yeast and fungi, a cholestanol analog with a 6-membered aza ring as side chain (22,26-azasterol) and 24-(R,S),25-epiminolanosterol, as well as combinations of these compounds with the C14 demethylase inhibitor ketoconazole. Both sterol analogs produced a dose-dependent reduction in the incorporation of radioactivity from [methyl-14C]methionine with IC50 values of 640 nM and 70 nM for 22,26-azasterol and 24,25-(R,S)-epiminolanosterol, respectively, indicating a specific inhibition of 24(25)-SMT. Correspondingly, it was found that the sterols present in control cells (ergosterol, 24-ethylcholesta-5,7,22-trien-3 beta-ol and precursors) were almost completely replaced by zymosterol (cholesta-8,24-dien-3 beta-ol) or a mixture of zymosterol, cholesta-7,24-dien-3 beta-ol and cholesta-5,7,24-trien-3 beta-ol when the parasites were exposed to the minimal growth inhibitory concentrations of 22,26-azasterol and 24-(R,S),25-epiminolanosterol, respectively. At sub-optimal concentrations of the inhibitors a complete disappearance of the 24-ethyl sterols was observed and a concomitant increase in the proportion of 24-methyl sterols, particularly delta 24(24') sterols. This showed that in T. cruzi the second methenylation step (catalyzed by delta 24(24') sterol methyl transferase) was significantly more sensitive to these inhibitors than the first and that the sterol analogs were also powerful inhibitors of the delta 24(24') sterol reductase. In growth-arrested epimastigotes resulting from their treatment with low (1-3 microM) concentrations of either sterol analog combined with sub optimal (100-300 nM) levels of ketoconazole the main sterol was lanosterol with no evidence 24-methylenedihydrolanosterol, the main sterol found in cells treated with growth inhibitory concentrations of the azole alone. Taken together, these results indicated that 24-alkyl sterols are essential growth factors for T. cruzi and that the preferred substrate of the delta 24(25) sterol methyl transferase in this organism is zymosterol.

Inhibition of phosphatidylcholine biosynthesis and cell proliferation in Trypanosoma cruzi by ajoene, an antiplatelet compound isolated from garlic.

Ajoene [(E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide], a potent antiplatelet compound derived from garlic, inhibits the proliferation of both epimastigotes and amastigotes of Trypanosoma cruzi, the causative agent of Chagas' disease. The growth of the epimastigote form was immediately arrested by 80 microM ajoene, while 100 microM induced cell lysis in 24 hr. In the amastigote form proliferating inside VERO cells, 40 microM ajoene was sufficient to eradicate the parasite from the host cells in 96 hr. Growth inhibition of the epimastigotes was accompanied by a gross alteration of the phospholipid composition of the treated cells in which phosphatidylcholine (PC), the major phospholipid class present in control cells, dropped to the least abundant phospholipid in cells treated with 60 microM ajoene for 96 hr, while its immediate precursor, phosphatidylethanolamine (PE), became the predominant species; this was correlated with a marked drop in the incorporation of [14C-U]acetate in PC and a corresponding increase in PE. Concomitant with the change in the phospholipid headgroup composition of the cells, the fatty acids esterified to this lipid fraction underwent a dramatic alteration due to the increase in the content of saturated fatty acids and a marked reduction in the content of linoleic (18:2) acid, which is the predominant fatty acid in control cells. We also found that ajoene inhibited the de novo synthesis of neutral lipids and, in particular, of sterols in the epimastigotes, but the resultant changes in the sterol composition were not sufficient to explain the antiproliferative effects of the drug. Electron-microscopy showed a concentration-dependent alteration of intracellular membranous structures, particularly the mitochondrion and endoplasmatic reticulum. The results suggest that one important factor associated with the antiproliferative effects of ajoene against T. cruzi is its specific alteration of the phospholipid composition of these cells.

Mevinolin (lovastatin) potentiates the antiproliferative effects of ketoconazole and terbinafine against Trypanosoma (Schizotrypanum) cruzi: in vitro and in vivo studies.

We have studied the antiproliferative effects of mevinolin (lovastatin), an inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, on the protozoan parasite Trypanosoma (Schizotrypanum) cruzi and its ability to potentiate the action of specific ergosterol biosynthesis inhibitors, such as ketoconazole and terbinafine, both in vitro and in vivo. Against the epimastigote form in vitro, mevinolin produced a dose-dependent reduction of the growth rate up to 25 microM, but at 50 and 75 microM, complete growth arrest and cell lysis took place after 144 and 96 h, respectively. A systematic study of the effects of mevinolin combined with ketoconazole and terbinafine, which act at different points in the ergosterol biosynthesis pathway, on the proliferation of epimastigotes indicated a synergic action, as shown by concave isobolograms and fractional inhibitory concentration indexes ranging from 0.17 to 0.54. Analysis of the sterol composition and de novo sterol synthesis in control and treated cells by thin-layer and gas-liquid chromatographies showed that the antiproliferative effects of the drug alone and in combination were correlated with the depletion of the endogenous ergosterol pool and particularly with a critical (exogenous) cholesterol/endogenous 4-desmethyl sterol ratio in the cells. When we studied the effects of mevinolin on the amastigote form proliferating inside Vero cells in vitro, only very modest effects on the parasites were observed up to 0.75 microM; above this concentration, significant deleterious effects on the host cells were found. However, when the same concentration of the drug was combined with ketoconazole, it was able to reduce by a factor of 10 the concentration of the azole required to eradicate the parasite (from 10 to 1 nM), again indicating a synergic action. On the other hand, a combination of mevinolin and terbinafine had only additive effects on amastigotes, but a ternary combination of mevinolin, ketoconazole, and terbinafine was again clearly synergistic. In vivo studies with a murine model of Chagas' disease showed that mevinolin can also potentiate the therapeutic effects of ketoconazole in this system; combined treatment with the two drugs at doses that alone offered only limited protection against the parasite was able to essentially eliminate circulating parasites and produce complete protection against death. These results confirm the synergic action against the proliferative stages of T. cruzi both in vitro and in vivo and in vivo of combined ergosterol biosynthesis inhibitors that act at different points in the pathway and suggest that mevinolin combined with azoles, such as ketoconazole, can be used in the treatment of human Chagas' disease.

Exercise and physical activity of adolescents.

Multiple studies point toward the beneficial role of regular exercise in preventing cardiovascular disease. The purpose of this study is to determine the prevalence of physical activity and exercise among adolescents from the only high school in Cataño, Puerto Rico. An open-ended, multiple choice questionnaire was administered to 106 students who were randomly chosen. Questions included frequency of exercise, type of sports which they practiced and would like to practice. Results were statistically analyzed. The study revealed that 53% of the participants did not exercise regularly, exercising less than once a week. Those who did exercise were involved in a competitive team sport (35% of females: volleyball; 65.8% of males: basketball). Approximately 1/3 of the females and of the males replied they would like to practice noncompetitive sports which should be incorporated into their lifestyles through early intervention in the school, home and through the primary care physician.