Antiprotozoal Activity of Benzoylthiourea Derivatives against Trypanosoma cruzi: Insights into Mechanism of Action.

For decades, only two nitroheterocyclic drugs have been used as therapeutic agents for Chagas disease. However, these drugs present limited effectiveness during the chronic phase, possess unfavorable pharmacokinetic properties, and induce severe adverse effects, resulting in low treatment adherence. A previous study reported that N-(cyclohexylcarbamothioyl) benzamide (BTU-1), N-(tert-butylcarbamothioyl) benzamide (BTU-2), and (4-bromo-N-(3-nitrophenyl) carbamothioyl benzamide (BTU-3) present selective antiprotozoal activity against all developmental forms of Trypanosoma cruzi Y strain. In this study, we investigated the mechanism of action of these compounds through microscopy and biochemical analyses. Transmission electron microscopy analysis showed nuclear disorganization, changes in the plasma membrane with the appearance of blebs and extracellular arrangements, intense vacuolization, mitochondrial swelling, and formation of myelin-like structures. Biochemical results showed changes in the mitochondrial membrane potential, reactive oxygen species content, lipid peroxidation, and plasma membrane fluidity. In addition, the formation of autophagic vacuoles was observed. These findings indicate that BTU-1, BTU-2, and BTU-3 induced profound morphological, ultrastructural, and biochemical alterations in epimastigote forms, triggering an autophagic-dependent cell death pathway.

Proteomic identification of metabolic changes in Paracoccidioides brasiliensis induced by a nitroheteroarylchalcone.

Aim: To access the metabolic changes caused by a chalcone derivative (LabMol-75) through a proteomic approach. Methods: Proteomic analysis was performed after 9 h of Paracoccidioides brasiliensis yeast (Pb18) cell incubation with the LabMol-75 at MIC. The proteomic findings were validated through in vitro and in silico assays. Results: Exposure to the compound led to the downregulation of proteins associated with glycolysis and gluconeogenesis, ß-oxidation, the citrate cycle and the electron transport chain. Conclusion: LabMol-75 caused an energetic imbalance in the fungus metabolism and deep oxidative stress. Additionally, the in silico molecular docking approach pointed to this molecule as a putative competitive inhibitor of DHPS.

Synergistic antifungal interaction of N-(butylcarbamothioyl) benzamide and amphotericin B against Cryptococcus neoformans.

Introduction: Cryptococcus neoformans is one of the leading causes of invasive fungal infections worldwide. Cryptococcal meningoencephalitis is the main challenge of antifungal therapy due to high morbidity and mortality rates, especially in low- and middle-income countries. This can be partly attributed to the lack of specific diagnosis difficulty accessing treatment, antifungal resistance and antifungal toxicity. Methods: In the present study, the effect of the synthetic thiourea derivative N-(butylcarbamothioyl) benzamide (BTU-01), alone and combined with amphotericin B (AmB), was evaluated in planktonic and sessile (biofilm) cells of C. neoformans. Results: BTU-01 alone exhibited a fungistatic activity with minimal inhibitory concentrations (MICs) ranging from 31.25 to 62.5 µg/mL for planktonic cells; and sessile MICs ranging from 125.0 to 1000.0 µg/mL. BTU-01 caused a concentration-dependent inhibitory activity on cryptococcal urease and did not interfere with plasma membrane fluidity. Molecular docking was performed on Canavalia ensiformis urease, and BTU-01 showed relevant interactions with the enzyme. The combination of BTU-01 and AmB exhibited synergistic fungicidal activity against planktonic and sessile cells of C. neoformans. Microscopic analysis of C. neoformans treated with BTU-01, alone or combined with AmB, revealed a reduction in cell and capsule sizes, changes in the morphology of planktonic cells; a significant decrease in the number of cells within the biofilm; and absence of exopolymeric matrix surrounding the sessile cells. Neither hemolytic activity nor cytotoxicity to mammalian cells was detected for BTU-01, alone or combined with AmB, at concentrations that exhibited antifungal activity. BTU-01 also displayed drug-likeness properties. Conclusion: These results indicate the potential of BTU-01, for the development of new strategies for controlling C. neoformans infections.

Paracoccidioides brasiliensis plasma membrane characterization by EPR spectroscopy and interactions with amphotericin B, miltefosine and nerolidol.

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to characterize the interactions of amphotericin B (AmB), miltefosine (MIL) and nerolidol (NER) with the plasma membrane of Paracoccidioides brasiliensis. Spin-labeled analogs of stearic acid and steroid androstane distributed into the plasma membrane of the fungus treated with AmB, showed strong interactions with putative AmB/sterol complexes. The observed increase in the EPR parameter 2A// caused by AmB can be interpreted as a remarkable reduction in the spin label mobility and/or an increase in the local polarity. The 2A// parameter reduced gradually as the concentration of MIL and NER increased. The membrane-water partition coefficient (KM/W) of the three compounds under study was estimated based on the minimum concentration of the compounds that causes a change in EPR spectrum. The KM/W values indicated that the affinity of the compounds for the P. brasiliensis membrane follows the order: AmB > MIL > NER. The minimum inhibitory concentration (MIC) values were lower than the respective minimum concentrations of the compounds to cause a change in the EPR spectrum, being ∼3.5-fold lower for AmB, 3.9-fold for MIL and ∼1.4-fold for NER. Taken together, the EPR spectroscopy results suggest that the anti-proliferative effects of the three compounds studied are associated with alterations in cell membranes. One of the most likely consequences of these changes would be electrolyte leakage.Communicated by Ramaswamy H. Sarma.

Proteomic alterations in Paracoccidioides brasiliensis caused by exposure to curcumin.

Paracoccidioides spp. are the etiological agent of paracoccidioidomycosis, a disease that causes skin lesions and affect the lungs and other organs. The current management of the disease is long and has several side effects that often lead the patient to give up the treatment, sequelae and even death. The search for new forms of treatment that minimize these drawbacks is very important. Thus, natural compounds are targets of great interest. Curcumin is one of the main components of the tubers of Curcuma longa, presenting medicinal effects well described in the literature, including the antifungal effect on Paracocidioides brasiliensis. Nevertheless, the mechanisms related to the antifungal effect of such compound are still unknown, so the objective of the present research is to understand what changes occur in the metabolism of P. brasiliensis after exposure to curcumin and to identify the main targets of the compound. Proteomic analysis as based on nanoUPLC-MS analysis and the functional classification of the identified proteins. The main metabolic processes that were being regulated were biologically validated through assays such as fluorescence microscopy, EPR and phagocytosis. Proteomic analysis revealed that curcumin regulates several metabolic processes of the fungus, including important pathways for energy production, such as the glycolytic pathway, beta oxidation and the glyoxylate cycle. Protein synthesis was down-regulated in fungi exposed to curcumin. The electron transport chain and the tricarboxylic acid cycle were also down-regulated, indicating that both the mitochondrial membrane and the mitochondrial activity were compromised. Plasma membrane and cell wall structure were altered following exposure to the compound. The fungus' ability to survive the phagocytosis process by alveolar macrophages was reduced. Thus, curcumin interferes with several metabolic pathways in the fungus that causes paracoccidioidomycosis. BIOLOGICAL SIGNIFICANCE: The challenges presented by the current treatment of paracoccidioidomycosis often contributing to patients' withdrawal from treatment, leading to sequelae or even death. Thus, the search for new treatment options against this disease is growing. The discovery that curcumin is active against Paracoccidioides was previously reported by our study group. Here, we clarify how the compound acts on the fungus causing its growth inhibition and decreased viability. Understanding the mechanisms of action of curcumin on P. brasiliensis elucidates how we can seek new alternatives and which metabolic pathways and molecular targets we should focus on in this incessant search to bring the patient a treatment with fewer adverse effects.

Ivermectin and curcumin cause plasma membrane rigidity in Leishmania amazonensis due to oxidative stress.

Spin label electron paramagnetic resonance (EPR) spectroscopy was used to study the mechanisms of action of ivermectin and curcumin against Leishmania (L.) amazonensis promastigotes. EPR spectra showed that treatment of the parasites with both compounds results in plasma membrane rigidity due to oxidative processes. With the IC50 and EPR measurements for assays using different parasite concentrations, estimations could be made for the membrane-water partition coefficient (KM/W), and the concentration of the compound in the membrane (cm50) and in the aqueous phase (cw50), which inhibits cell growth by 50%. The KM/W values indicated that ivermectin has a greater affinity than curcumin for the parasite membrane. Therefore, the activity of ivermectin was higher for experiments with low cell concentrations, but for concentrations greater than 1.5 × 108 parasites/mL the compounds did not show significantly different results. The cm50 values indicated that the concentration of compound in the membrane leading to growth inhibition or membrane alteration is approximately 1 M for both ivermectin and curcumin. This high membrane concentration suggests that many ivermectin molecules per chlorine channel are needed to cause an increase in chlorine ion influx.

Mycobacterium abscessus cell wall and plasma membrane characterization by EPR spectroscopy and effects of amphotericin B, miltefosine and nerolidol.

Spin label electron paramagnetic resonance (EPR) spectroscopy was used to characterize the components of the Mycobacterium abscessus massiliense cell envelope and their interactions with amphotericin B (AmB), miltefosine (MIL), and nerolidol (NER). Spin labels analogous to stearic acid and phosphatidylcholine (PC) were distributed on an envelope layer with fluidity comparable to other biological membranes, probably the mycobacterial cell wall, because after treatment with AmB a highly rigid spectral component was evident in the EPR spectra. Methyl stearate analogue spin labels found a much more fluid membrane and did not detect the presence of AmB, except for at very high drug concentrations. Unlike other spin-labeled PCs, the TEMPO-PC spin probe, with the nitroxide moiety attached to the choline of the PC headgroup, also did not detect the presence of AmB. On the other hand, the steroid spin labels were not distributed across the membranes of M. abscessus and, instead, were concentrated in some other location of the cell envelope. Both MIL and NER compounds at 10 µM caused increased fluidity in the cell wall and plasma membrane. Furthermore, NER was shown to have a remarkable ability to extract lipids from the mycobacterial cell wall. The EPR results suggest that the resistance of mycobacteria to the action of AmB must be related to the fact that this drug does not reach the bacterial plasma membrane.

Plasma membrane rigidity effects of 4-hydroxy-2-nonenal in Leishmania, erythrocyte and macrophage.

4-hydroxy-2-nonenal (HNE) is a reactive aldehyde produced by cells under conditions of oxidative stress, which has been shown to react with proteins and phosphatidylethanolamine in biological membranes. Using electron paramagnetic resonance (EPR) spectroscopy of a spin label it was demonstrated that 2 h of treatment with HNE causes membrane rigidity in promastigotes of Leishmania (L.) amazonensis, J774.A1 macrophages and erythrocytes. Remarkable fluidity-reducing effects on the parasite membrane were observed at HNE concentrations approximately 4-fold lower than in the case of erythrocyte and macrophage membranes. Autofluorescence of the parasites in PBS suspension (1 × 107 cell/mL) with excitation at 354 nm showed a linear increase of intensity in the range of 400 to 600 nm over 3 h after treatment with 30 µM HNE. Parasite ghosts prepared after this period of HNE treatment showed a high degree of membrane rigidity. Bovine serum albumin (BSA) in PBS treated with HNE for 2 h showed an increase in molecular dynamics and suffered a decrease in its ability to bind a lipid probe. In addition, the antiproliferative activity of L. amazonensis promastigotes, macrophage cytotoxicity and hemolytic potential were assessed for HNE. An IC50 of 24 µM was found, which was a concentration > 10 times lower than the cytotoxic and hemolytic concentrations of HNE. These results indicate that the action of HNE has high selectivity indices for the parasite as opposed to the macrophage and erythrocyte.

Combination of lipid nanoparticles and iontophoresis for enhanced lopinavir skin permeation: Impact of electric current on lipid dynamics.

Nanostructured lipid carriers (NLC)-loaded with lopinavir (LPV) were developed for its iontophoretic transdermal delivery. Electronic paramagnetic resonance (EPR) spectroscopy of fatty acid spin labels and differential scanning calorimetry (DSC) were applied to investigate the lipid dynamic behavior of NLC before and after the electrical current. In vitro release and permeation studies, with and without anodic and cathodic iontophoresis were also performed. NLC-LPV had nanometric size (179.0 ± 2.5 nm), high drug load (∼x223C 4.14%) and entrapment efficiency (EE) (∼x223C 80%). NLC-LPV was chemically and physically stable after applying an electric current. The electrical current reduced EE after 3 h (67.21 ± 2.64%), resulting in faster LPV in vitro release. EPR demonstrated that iontophoresis decreased NLC lipid dynamics, which is a long-lasting effect. DSC studies demonstrated that electrical current could trigger the polymorphic transition of NLC and drug solubilization in the lipid matrix. NLC-LPV, combined with iontophoresis, allowed drug quantification in the receptor medium, unlike unloaded drugs. Cathodic iontophoresis enabled the quantification of about 7.9 µg/cm2 of LPV in the receptor medium. Passive NLC-LPV studies had to be done for an additional 42 h to achieve similar concentrations. Besides, anodic iontophoresis increased by 1.8-fold the amount of LPV in the receptor medium, demonstrating a promising antiviral therapy strategy.

Comparative EPR spectroscopy analysis of amphotericin B and miltefosine interactions with Leishmania, erythrocyte and macrophage membranes.

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to study the interactions of amphotericin B (AmB) with the plasma membrane of Leishmania (L.) amazonensis promastigotes, human erythrocytes and J774.A1 murine macrophages, in comparison with reported and novel data for miltefosine (MIL). One of the objectives of this work is to look for the relationships between the activities of these two drugs in the Leishmania parasite with their changes in the cell membrane. A spin-labeled stearic acid inserted into the cell membranes showed strong interactions with putative AmB/sterol complexes, characterized by reductions in molecular dynamics. The concentration of the drugs in the plasma membrane that reduced the cell population by 50%, and the membrane-water partition coefficient of the drugs, were assessed. These biophysical parameters enabled estimates of possible therapeutic concentrations of these two drugs in the interstitial fluids of the tissues to be made. AmB displayed higher affinity for the plasma membrane of L. amazonensis than for that of the macrophage and erythrocyte, denoting a preference for a membrane that contains ergosterol. AmB also demonstrated higher hemolytic potential than MIL for measurements on erythrocytes in both PBS and whole blood. For MIL, the EPR technique detected membrane changes induced by the drug in the same concentration range that inhibited the growth of parasites, but in the case of AmB, an 8-fold higher concentration of the IC50 was necessary to observe a reduction in membrane fluidity, suggesting a better localized effect of AmB on the membrane. Taken together, the results demonstrate that the antiproliferative and cytotoxic effects of both drugs are associated with changes in cell membranes.

Membrane dynamics in Leishmania amazonensis and antileishmanial activities of ß-carboline derivatives.

Two ß-carboline compounds, 8i and 6d, demonstrated in vitro antileishmanial activity against Leishmania (L.) amazonensis promastigotes similar to that of miltefosine (MIL). Estimates of the membrane-water partition coefficient (KM/W) and the compound concentrations in the membrane (cm50) and aqueous phase (cw50) for half maximal inhibitory concentration were made. Whereas these biophysical parameters for 6d were not significantly different from those reported for MIL, 8i showed lower affinity for the parasite membrane (lower KM/W) and a lower concentration of the compound in the membrane required to inhibit the growth of the parasite (lower cm50). A 2-hour treatment of Leishmania promastigotes with the compounds 8i and 6d caused membrane rigidity in a concentration-dependent manner, as demonstrated by the electron paramagnetic resonance (EPR) technique and spin label method. This increased rigidity of the membrane was interpreted to be associated with the occurrence of cross-linking of oxidized cytoplasmic proteins to the parasite membrane skeleton. Importantly, the two ß-carboline-oxazoline derivatives showed low hemolytic action, both in experiments with isolated red blood cells or with whole blood, denoting their great Leishmania/erythrocyte selectivity index. Using electron microscopy, changes in the membrane of both the amastigote and promastigote form of the parasite were confirmed, and it was demonstrated that compounds 8i and 6d decreased the number of amastigotes in infected murine macrophages. Furthermore, 8i and 6d were more toxic to the protozoa than to J774A.1 macrophages, with treated promastigotes exhibiting a decrease in cell volume, mitochondrial membrane potential depolarization, accumulation of lipid bodies, increased ROS production and changes in the cell cycle.

Analysis of the Interactions of Amphotericin B with the Leishmania Plasma Membrane Using EPR Spectroscopy.

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to study the interactions of amphotericin B (AmB) with the plasma membrane of Leishmania amazonensis promastigotes, erythrocytes, and J774 macrophages. Spin labels embedded into the cell membranes detected strong interactions with putative AmB/sterol complexes that resulted in pronounced changes in the EPR spectra, which can be interpreted as a reduction in membrane fluidity or an increase in the polarity assessed by the spin probe. The EPR spectra of spin-labeled lipids corroborated the findings that AmB does not enter phospholipid membrane-sterol models and probably forms extramembranous aggregates, as predicted by the sterol sponge model. Furthermore, these aggregates were shown to extract the spin probe androstanol from the lipid bilayer. However, in contrast to the results for the model membrane, EPR spectroscopy suggested that AmB easily enters the membranes of the studied cells, implying that the entry process is dependent on interactions with the membrane proteins.

Antileishmanial activity of the chalcone derivative LQFM064 associated with reduced fluidity in the parasite membrane as assessed by EPR spectroscopy.

A novel chalcone derivative, LQFM064, demonstrated antileishmanial activity against Leishmania (L.) amazonensis, with an IC50 value of ~10 µM for the promastigote form. Electron paramagnetic resonance (EPR) spectroscopy of a spin-labeled stearic acid incorporated in the plasma membrane of L. amazonensis promastigotes revealed that after 2 h of treatment with LQFM064, the parasite showed remarkable reductions in membrane fluidity. The features of the altered EPR spectra were similar to those reported for the erythrocyte membrane, which was suggested to be due to the cross-linking of oxidized hemoglobin with the cytoskeleton spectrin. In comparison to miltefosine (MIL), LQFM064 demonstrated a much lower hemolytic potential against both erythrocytes in PBS and whole blood, less cytotoxicity in J774.A1 macrophages and equivalent ability to kill parasites internalized in J774.A1 macrophages. Measurements of the IC50 values for assays with different cell concentrations enabled the estimation of the membrane-water partition coefficient (KM/W), as well as the concentrations of LQFM064 in membrane (cm50) and aqueous phase (cw50) that reduces the cell population by 50%. From the KM/W and cm50 values it was deduced that LQFM064 has a greater affinity than MIL for the parasite membrane, but the antiproliferative activity of both substances is exerted at a similar concentration in the plasma membrane.

Effects of bovine serum albumin (BSA) on the excited-state properties of meso-tetrakis(sulfonatophenyl) porphyrin (TPPS4).

To infer changes in the photophysical properties of porphyrins due to complexation with albumin, a combination of Z-scan and conventional spectroscopic techniques was employed. We measured the characteristics of excited states of meso-tetrakis(sulfonatophenyl) porphyrin bound to bovine serum albumin and observed that the binding reduces the intersystem crossing quantum yield and increases the internal conversion one. A reverse saturable absorption process was observed in the nanosecond timescale. These results are important for prediction of the efficiency of this complex in medical and optical applications, because associating porphyrins to proteins enables better accumulation in tumors and improves its stability in optical devices, but at the same time, decreases its triplet quantum yield.

Antileishmanial and cytotoxic activities of ionic surfactants compared to those of miltefosine.

Using the electron paramagnetic resonance (EPR) of spin-labeled stearic acid and a spin label chemically attached to the membrane proteins, the interaction of miltefosine (MIL) and the ionic surfactants sodium dodecyl sulfate (SDS, anionic), cetyltrimethylammonium chloride (CTAC, cationic) and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS, zwitterionic) with the plasma membrane of Leishmania (L.) amazonensis promastigotes was studied. The spin-label EPR data indicated that the four compounds studied have the ability to increase the molecular dynamics of membrane proteins to a large extent. Compared to the other compounds, SDS produced the smallest increases in dynamics and demonstrated the lowest antileishmanial activity and cytotoxicity to J774.A1 macrophages. The activities of the other three compounds were not different from each other, but CTAC had a stronger activity against L. amazonensis promastigotes at higher cellular concentrations (> 1 × 109 cells/mL) and was the most effective against L. amazonensis-infected macrophages. However, CTAC was also the most cytotoxic to macrophages. By measuring the IC50/CC50 values for assays of different cell concentrations, we estimated the membrane-water partition coefficient (KM/W) as well as the concentrations in the membrane (cm50) and aqueous phase (cw50) of the compounds at their IC50/CC50. Compared to the other compounds, SDS showed the lowest value of KM/W and the highest value of cm50. In all experiments in this study, the data for the zwitterionic molecules HPS and MIL were not significantly different.

Interactions of miltefosine with erythrocyte membrane proteins compared to those of ionic surfactants.

For miltefosine (MIL), a zwitterionic alkylphospholipid approved for leishmaniasis treatment, the mechanism of action is not well established. Electron paramagnetic resonance (EPR) spectroscopy has indicated that the interaction of MIL with membrane proteins has similarities to that of ionic surfactants. A general concern about leishmanicides is their high hemolytic potential, so we decided to compare the interactions of MIL and three ionic surfactants with the erythrocyte membrane. Measurements with two different spin labels indicated that the surfactants sodium dodecyl sulfate (SDS, anionic), cetyltrimethylammonium chloride (CTAC, cationic) and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS, zwitterionic) as well as MIL increase the dynamics of erythrocyte membrane proteins in a concentration-dependent manner. SDS produced the smallest increases in protein dynamics and was also the least hemolytic for measurements in PBS and in whole blood. Spin label EPR measurements performed directly in the blood plasma detected increased albumin stiffness caused by 2.5 mM SDS due to electrostatic/hydrophobic interactions. For 10 mM concentrations of the compounds, the EPR spectra showed a fraction of albumin with greater mobility and another with the same as that of the untreated plasma. The zwitterionic compounds MIL and HPS did not present significant differences in this study.

In vitro antileishmanial and cytotoxic activities of nerolidol are associated with changes in plasma membrane dynamics.

The sesquiterpene nerolidol is a membrane-active compound that has demonstrated antitumor, antibacterial, antifungal and antiparasitic activities. In this study, we used electron paramagnetic resonance (EPR) spectroscopy and biophysical parameters determined via cell culture assays to study the mechanisms underlying the in vitro antileishmanial activity of nerolidol. The EPR spectra of a spin-labeled stearic acid indicated notable interactions of nerolidol with the cell membrane of Leishmania amazonensis amastigotes. The nerolidol IC50 values in L. amazonensis amastigotes and promastigotes were found to depend on the cell concentration used in the assay. This dependence was described by an equation that considers various cell suspension parameters, such as the 50% inhibitory concentrations of nerolidol in the cell membrane (cm50) and the aqueous phase (cw50) and the membrane-water partition coefficient of nerolidol (KM/W). Via cytotoxicity (CC50) and hemolytic potential (HC50) data, these parameters were also determined for nerolidol in macrophages and erythrocytes. With a cw50 of 125 µM, macrophages were less sensitive to nerolidol than amastigotes and promastigotes, which had mean cw50 values of 56 and 74 µM, respectively. The estimated cm50 values of nerolidol for amastigotes and promastigotes and macrophages were between 2.6 and 3.0 M, indicating substantial accumulation of nerolidol in the cell membrane. In addition, the spin-label EPR data indicated that membrane dynamic changes occurred in L. amazonensis amastigotes at concentrations similar to the nerolidol IC50 value.

Photodynamic inactivation of Bovine herpesvirus type 1 (BoHV-1) by porphyrins.

In this work, the photodynamic efficiency of anionic meso-tetrakis sulfonophenyl (TPPS4), cationic meso-tetrakis methylpyridiniumyl (TMPyP) and their zinc complexes (ZnTPPS4 and ZnTMPyP) in the inactivation of Bovine herpesvirus type 1 (BoHV-1) was evaluated. At a non-cytotoxic concentration, all porphyrins showed significant antiviral activity after irradiation using a halogen lamp. The efficiency of the cationic porphyrins was higher than that of the anionic ones. Porphyrin complexation with zinc increases its lipophilicity and the number of absorbed photons, dramatically reducing the time for complete virus inactivation. The high superposition of the compound optical absorption and light source emission spectra played a key role in the virus inactivation efficiency. The results demonstrated the high effectivity of the photodynamic inactivation of BoHV-1. This method can be used as an auxiliary in the treatment of disorders attributed to BoHV-1 infection, and the porphyrins are promising photosensitizers for this application.

Photodynamic evaluation of tetracarboxy-phthalocyanines in model systems.

The present work reports the synthesis, photophysical and photochemical characterization and photodynamic evaluation of zinc, aluminum and metal free-base tetracarboxy-phthalocyanines (ZnPc, AlPc and FbPc, respectively). To evaluate the possible application of phthalocyanines as a potential photosensitizer the photophysical and photochemical characterization were performed using aqueous (phosphate-buffered solution, PBS) and organic (dimethyl sulfoxide, DMSO) solvents. The relative lipophilicity of the compounds was estimated by the octanol-water partition coefficient and the photodynamic activity evaluated through the photooxidation of a protein and photohemolysis. The photooxidation rate constants (k) were obtained and the hemolytic potential was evaluated by the maximum percentage of hemolysis achieved (Hmax) and the time (t50) to reach 50% of the Hmax. Although these phthalocyanines are all hydrophilic and possess very low affinity for membranes (log PO/W=-2.0), they led to significant photooxidation of bovine serum albumin (BSA) and photohemolysis. Our results show that ZnPc was the most efficient photosensitizer, followed by AlPc and FbPc; this order is the same as the order of the triplet and singlet oxygen quantum yields (ZnPc>AlPc>FbPc). Furthermore, together, the triplet, fluorescence and singlet oxygen quantum yields of zinc tetracarboxy-phthalocyanines suggest their potential for use in theranostic applications, which simultaneously combines photodiagnosis and phototherapy.

Hemolytic potential of miltefosine is dependent on cell concentration: Implications for in vitro cell cytotoxicity assays and pharmacokinetic data.

Miltefosine possesses antiparasitic, antibacterial, antifungal and antitumor activities; however, its mechanism of action is not well established. In the current work, the miltefosine concentrations required to achieve 50% hemolysis in PBS were shown to vary from 600 µM using 5×10(9) cells/mL to ~2.9 µM for ~5×10(6) cells/mL. This cell concentration-dependent hemolytic potential was described using an equation that included the membrane-water partition coefficient (K) and miltefosine concentrations in the cell membrane (cm) and aqueous medium (cw) as variables. The best-fit values for the 50% hemolysis data were log K=4.68, cm=110.8 mM, and cw=2.3 µM. Hemolysis measurements in whole blood were used to determine the erythrocyte membrane-plasma partition coefficient of miltefosine (Log KM/P=1.77). Additionally, miltefosine concentration in whole blood was found to be ~86% of that in plasma. Previously reported clinical pharmacokinetics data indicate that the plasma concentration of miltefosine peaks at ~90 µg/mL when treating visceral leishmaniasis. Using this concentration, which corresponds to ~77 µg/mL miltefosine in whole blood, we found only 2.8% hemolysis. Significant hemolysis (5.4%) was observed only after doubling the concentration to 180 µg/mL. Recently reported data indicate that miltefosine inhibitory concentrations in Leishmania are also dependent on cell concentration. The biophysical parameters assessed in the current study indicated that this type of response is associated with the accumulation of the drug in the cell membrane, which becomes damaged when critical drug concentrations are reached.