LyeTx I, a potent antimicrobial peptide from the venom of the spider Lycosa erythrognatha.

LyeTx I, an antimicrobial peptide isolated from the venom of Lycosa erythrognatha, known as wolf spider, has been synthesised and its structural profile studied by using the CD and NMR techniques. LyeTx I has shown to be active against bacteria (Escherichia coli and Staphylococcus aureus) and fungi (Candida krusei and Cryptococcus neoformans) and able to alter the permeabilisation of L: -alpha-phosphatidylcholine-liposomes (POPC) in a dose-dependent manner. In POPC containing cholesterol or ergosterol, permeabilisation has either decreased about five times or remained unchanged, respectively. These results, along with the observed low haemolytic activity, indicated that antimicrobial membranes, rather than vertebrate membranes seem to be the preferential targets. However, the complexity of biological membranes compared to liposomes must be taken in account. Besides, other membrane components, such as proteins and even specific lipids, cannot be discarded to be important to the preferential action of the LyeTx I to the tested microorganisms. The secondary structure of LyeTx I shows a small random-coil region at the N-terminus followed by an alpha-helix that reached the amidated C-terminus, which might favour the peptide-membrane interaction. The high activity against bacteria together with the moderate activity against fungi and the low haemolytic activity have indicated LyeTx I as a good prototype for developing new antibiotic peptides.

Determination of the osmotic active drug concentration in the cytoplasm of anthracycline-resistant and -sensitive K562 cells.

A fluorescence method was used to follow the interaction of 4'-o-tetrahydropyranyladriamycin (THP-ADR) with drug-resistant and -sensitive K562 cells. The amounts of drug bound to the nuclei at the steady state, Cn and at the equilibrium state, CN, once the membrane has been solubilized with Triton X-100, have been determined as a function of the pH outside the cells (pHe): Cn increased and CN decreased as pHe increased. At a given pH value outside the cells, CN is the same for both sensitive and resistant cells, whereas Cn is lower in resistant cells as compared to sensitive cells. Using the observation that the essential binding characteristics of THP-ADR in nuclei are the same for both types of cell, the osmotic active drug concentration, Ci, in the cytoplasm of the cells was determined at different values of pHe. Using fluorescent dye, the cytoplasmic pH was determined and found equal to 7.2 +/- 0.1 in both types of cell. In sensitive cells, the equilibrium transmembrane concentrations verified the relation [DH+]i/[DH+]e = [H +]i/[H+]e where [DH +]i and [DH +]e stand for the concentration of protonated form of the drug inside and outside the cells, respectively. This indicates that the uptake of the drug occurs through free permeation of the neutral form of the drug in response to delta pH gradient. Such a relation is not verified in the case of resistant cells.

Permeability of lipid bilayer to anthracycline derivatives. Role of the bilayer composition and of the temperature.

The uptake of three anthracycline derivatives: doxorubicin, daunorubicin and pirarubicin, into large unilamellar vesicles (LUV) in response to a driving force provided by DNA encapsulated inside the LUV has been investigated as a function of the temperature and of the bilayers lipid composition. The kinetics of the decay of the anthracycline fluorescence in the presence of DNA-containing liposome was used to follow the diffusion of the drug through the membrane. For the three drugs, the permeability coefficient of the neutral form of the drug (P0) decreases as the amount of negatively charged phospholipid in the bilayers increases. This can be explained by the fact that the kinetics of passive diffusion of the drugs depends on the amount of neutral form embedded in the polar head group region, which decreases as the quantity of negatively charged phospholipids increases. P0 also decreases as the amount of cholesterol, that makes the bilayer more rigid, increases. The activation energies, Ea, for the passage of the neutral form of these anthracyclines through the bilayers lie within 100 +/- 15 kJ x ml-1, except for pirarubicin and doxorubicin through anionic phospholipid-rich membranes (Ea = 57 kJ x mol-1) and cholesterol-rich membranes (Ea = 167 kJ x mol-1).

Comparison of the binding of anthracycline derivatives to purified DNA and to cell nuclei.

Fluorescence method was used to study the interactions of anthracyclines with purified DNA and with cell nuclei at 37 degrees C, at pH ranging from 6.8 to 8. Four anthracyclines were used; adriamycin (ADR), 4'-o-tetrahydropyranyladriamycin (THP-ADR), daunorubicin (DNR) and aclacinomycin (ACM). The values of pKa of deprotonation of these four drugs in the pH range 6.5-8.5 are 8.4, 7.7, 8.4 and 7.0 for ADR, THP-ADR, DNR and ACM, respectively. The overall binding constants K* of these four drugs to purified DNA was determined at various pH values. The binding constants K0 and K+ of the respectively neutral form and once protonated form of the drugs to DNA were calculated. Using cell nuclei from K562 cells, the amount of drug intercalated (CN) within the nuclei of K562 cells and the amount of free drug (CE) in the solution were determined at various pH values: measuring at the same pH values, a linear correlation occurred between K* and CN/CE.

Fluorinated phosphatidylcholine-based liposomes: H+/Na+ permeability, active doxorubicin encapsulation and stability, in human serum.

The active encapsulation of doxorubicin (DOX) into fluorinated liposomes, the stability of these liposomes with respect to encapsulated DOX release in buffer and in human serum, and their H+/Na+ membrane permeability have been investigated and compared to those of their conventional hydrogenated analogues. These fluorinated liposomes are made from highly fluorinated phosphatidylcholines and contain a fluorinated core within their membrane. We found that the presence of this fluorinated core is not a barrier for the active encapsulation of DOX. Efficient (> 90%) and stable loading could be achieved using a transmembrane ammonium sulfate or even, in the absence of Na+, a transmembrane pH gradient. The higher H+/Na+ permeability found for the fluorinated membranes, as compared to conventional ones, is responsible for the lower stability observed for the DOX-loaded fluorinated liposomes when incubated in a physiological buffer (PBS) or in human serum. It is also noticeable that the retention of DOX is increased in human serum and for the liposomes whose membranes are in a gel or in a semi-fluid semi-gel state at 37 degrees C.

Anthracycline incorporation in human lymphocytes. Kinetics of uptake and nuclear concentration.

Fluorescence emission spectra from anthracycline-treated cells suspended in buffer have been studied. The kinetics of uptake and the nuclear concentration of anthracyclines in human lymphocytes have thus been determined using the fluorescence properties of these drugs. Four anthracyclines have been used: adriamycin (ADR), 4'-O-tetrahydropyranyl-adriamycin (THP-ADR), carminomycin (CAR) and aclacinomycin A (ACM). We have shown that no quenching of the drug fluorescence is obtained through interaction of the drugs with the various components of the cell except the nucleus. No quenching is observed with cells lacking nucleus such as platelets and erythrocytes. Quenching of drug fluorescence occurs when drugs intercalate between base pairs of DNA only. This allows rapid determination of the amount of drug intercalated between nuclear base pairs in the steady state. We have thus estimated that one molecule of drug can bind for every 10, 8.3, 10 and 6.7 DNA base pairs in the case of ADR, THP-ADR, ACM and CAR, respectively. The kinetics of drug incorporation into the nucleus, once the cells have been solubilized with triton X-100, is very fast for the four drugs. However, the kinetics of drug uptake by the intacts cells strongly depends on the nature of the drug. When 10(9) cells/l are incubated with 1 microM drug, 50% of the final nuclear concentration is reached within 97 min, 3.2 min, 3.0 min and 1.2 min in the case of ADR, THP-ADR, CAR and ACM, respectively. The kinetics directly correlates with the polarity of the molecule.

Membrane-phorbol ester interactions monitored by circular dichroism.

The interaction of phorbol 12,13-dibutyrate (PDBu), 12-O-retinoylphorbol 13-acetate (RPA) and 12-O-tetradecanoylphorbol 13-acetate (TPA) with L-alpha-phosphatidylserine-containing small unilamellar vesicles or erythrocyte ghosts was monitored by circular dichroism (CD). No change in the CD spectra of PDBu was observed upon binding, while RPA and TPA spectra were slowly affected by the interaction. The changes in RPA and TPA spectra were assigned to the embedding of these molecules in the membrane bilayers. In the presence of 10(8) cells/ml, after one minute incubation, about 2 to 5% of the amount of phorbol ester added is embedded in the membrane. It is suggested that either phorbol esters entering the membrane is not a prerequisite for protein kinase C activation or the amount of phorbol esters necessary to activate protein kinase C is very small.

Permeability and stability in buffer and in human serum of fluorinated phospholipid-based liposomes.

The stability (with respect to encapsulated carboxyfluorescein release) of fluorinated liposomes and their membrane permeability have been investigated in buffer and in human serum as compared to conventional hydrogenated analogues. These fluorinated liposomes are made from highly fluorinated phosphatidylcholines and contain a fluorinated core within their membrane. In buffer and in their fluid state, the fluorinated liposomes retain much more efficiently their entrapped content and display lower membrane permeability coefficients than any of their hydrogenated counterparts. This indicates that the fluorinated core acts as a very efficient barrier to permeation. In terms of molecular structure/permeability relationships, the thicker the fluorinated lipophobic core, the more efficient the barrier to permeation. In their gel state, the fluorinated core has, however, almost no effect on permeation. Interestingly, some of the 'fluid' fluorinated liposomes were even less permeable than 'gel' or 'gel-like' ones, including egg phosphatidylcholines/cholesterol liposomes. Human serum destabilizes the 'fluid' fluorinated liposomes but to a lesser extent than the 'fluid' hydrogenated ones, indicating that the fluorinated lipophobic core inside the liposomal membrane protects the vesicles, possibly by reducing their interactions with serum components. 'Gel' or 'gel-like' fluorinated liposomes are significantly more stable in serum than in buffer. They are also more stable than conventional 'gel' or 'gel-like' liposomes.

Angiotensin-(1-7): blood, heart, and blood vessels.

In the past few years, there has been a growing interest in the heptapeptide Angiotensin(Ang)-(1-7), mainly because of its ability to counter regulate many of Ang II actions. Furthermore, heart and blood vessels are important target tissues for Ang-(1-7) formation and actions. The introduction of novel tools, such as the Ang-(1-7) antagonists, A-779 and D-pro7-Ang-(1-7), the Ang-(1-7) agonist AVE 0991, transgenic rats TGR(A-1-7)3292, and use of liposome-encapsulated Ang-(1-7) for evaluating the biochemical and functional role of Ang-(1-7), have produced a great impact in this field of research. Moreover, the recent identification of the Ang-(1-7)-forming enzyme ACE2 and of the Ang-(1-7) receptor Mas will allow important advances in our understanding of the physiological and pathological role of this peptide. In this review, we will discuss the current knowledge concerning the biological effects of Ang-(1-7) in the blood, heart, and blood vessels. In addition, we will highlight the possible applications of agonists of its receptor as therapeutic agents in cardiovascular and related diseases.

Pharmacokinetic and parasitological evaluation of the bone marrow of dogs with visceral leishmaniasis submitted to multiple dose treatment with liposome-encapsulated meglumine antimoniate.

The aim of the present study was to evaluate the impact of a multiple dose regimen of a liposomal formulation of meglumine antimoniate (LMA) on the pharmacokinetics of antimony in the bone marrow of dogs with visceral leishmaniasis and on the ability of LMA to eliminate parasites from this tissue. Dogs naturally infected with Leishmania chagasi received 4 intravenous doses of either LMA (6.5 mg antimony/kg body weight, N = 9), or empty liposomes (at the same lipid dose as LMA, N = 9) at 4-day intervals. A third group of animals was untreated (N = 8). Before each administration and at different times after treatment, bone marrow was obtained and analyzed for antimony level (LMA group) by electrothermal atomic absorption spectrometry, and for the presence of Leishmania parasites (all groups). There was a significant increase of antimony concentration from 0.76 microg/kg wet organ (4 days after the first dose) to 2.07 microg/kg (4 days after the fourth dose) and a half-life of 4 days for antimony elimination from the bone marrow. Treatment with LMA significantly reduced the number of dogs positive for parasites (with at least one amastigote per 1000 host cells) compared to controls (positive dogs 30 days after treatment: 0 of 9 in the LMA group, 3 of 9 in the group treated with empty liposomes and 3 of 8 in the untreated group). However, complete elimination of parasites was not achieved. In conclusion, the present study showed that multiple dose treatment with LMA was effective in improving antimony levels in the bone marrow of dogs with visceral leishmaniasis and in reducing the number of positive animals, even though it was not sufficient to achieve complete elimination of parasites.

Cardiovascular and behavioral effects produced by administration of liposome-entrapped GABA into the rat central nervous system.

Liposomes are nanosystems that allow a sustained release of entrapped substances. Gamma-aminobutyric acid (GABA) is the most prevalent inhibitory neurotransmitter of the central nervous system (CNS). We developed a liposomal formulation of GABA for application in long-term CNS functional studies. Two days after liposome-entrapped GABA was injected intracerebroventricularly (ICV), Wistar rats were submitted to the following evaluations: (1) changes in mean arterial pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA) to ICV injection of bicuculline methiodide (BMI) in anesthetized rats; (2) changes in cardiovascular reactivity to air jet stress in conscious rats; and (3) anxiety-like behavior in conscious rats. GABA and saline-containing pegylated liposomes were prepared with a mean diameter of 200 nm. Rats with implanted cannulas targeted to lateral cerebral ventricle (n = 5-8/group) received either GABA solution (GS), empty liposomes (EL) or GABA-containing liposomes (GL). Following (48 h) central microinjection (2 µL, 0.09 M and 99 g/L) of liposomes, animals were submitted to the different protocols. Animals that received GL demonstrated attenuated response of RSNA to BMI microinjection (GS 48 ± 9, EL 43 ± 9, GL 11 ± 8%; P < 0.05), blunted tachycardia in the stress trial (ΔHR: GS 115 ± 14, EL 117 ± 10, GL 74 ± 9 bpm; P<0.05) and spent more time in the open arms of elevated plus maze (EL 6 ± 2 vs. GL 18 ± 5%; P = 0.028) compared with GS and EL groups. These results indicate that liposome-entrapped GABA can be a potential tool for exploring the chronic effects of GABA in specific regions and pathways of the central nervous system.

Extended in vivo blood circulation time of fluorinated liposomes.

The clearance from blood circulation of fluorinated liposomes made with perfluoroalkylated phosphatidylcholines was investigated in mice using liposome-entrapped 5(6)-carboxyfluorescein. The presence of a fluorinated core inside the membrane strongly retards their blood clearance. The fluorinated vesicles showed circulation half-lives of up to 8.6 h, which are 6-13 and 3-6 times larger than those of similarly sized conventional distearoylphosphatidylcholine and distearoylphosphatidylcholine/cholesterol liposomes, respectively. Their blood clearance was similar to that of some polyethylene glycol (PEG)-labelled 'stealth' liposomes and was dose-independent in a 3.3-330 mumol/kg body weight dose range.

Energy-dependent efflux from Leishmania promastigotes of substrates of the mammalian multidrug resistance pumps.

We demonstrated the existence of three transport activities in promastigotes of Leishmania braziliensis, Leishmania guyanensis, and Leishmania mexicana. The first activity, an energy-dependent efflux of pirarubicin, was observed in all Leishmania species and inhibited by verapamil, by 2-[4-(diphenylmethyl)-1-piperazinyl]ethyl-5-(trans-4,6-dimethyl-1, 3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4-(3-nitrophenyl)-3-py ridinecarboxylate P oxide (PAK104P) and by the phenothiazine derivatives: thioridazine, prochlorperazine, trifluoperazine, chlorpromazine and trifluoropromazine. The second activity, an energy-dependent efflux of calcein acetoxymethylester, was observed in all Leishmania species and inhibited by PAK104P and the same phenothiazine derivatives, but not by verapamil. The third activity, an energy-dependent efflux of calcein, was clearly detected in L. braziliensis and guyanensis and inhibited only by prochlorperazine and trifluoperazine. The fact that prochlorperazine and trifluoperazine inhibited the energy-dependent efflux of the three substrates suggests that these activities are mediated by the same transport system. It is noteworthy that the transport system identified in this study shares several properties with the mammalian multidrug resistance pump, MRP1. Pirarubicin, calcein acetoxymethylester and calcein are well known substrates of the MRP. Furthermore, the three types of inhibitors are also inhibitors of the MRP function.

Encapsulation of native crotoxin in liposomes: a safe approach for the production of antivenom and vaccination against Crotalus durissus terrificus venom.

Crotoxin, the neurotoxic component of Crotalus durissus terrificus (Cdt) venom that displays phospholipase A2 activity, was successfully encapsulated into dehydration-rehydration vesicles (DRV/crotoxin) and reverse-phase evaporation vesicles (REV/crotoxin) made from sphingomyelin and cholesterol. The encapsulation efficiency of native crotoxin was higher in DRV/crotoxin than in REV/crotoxin. DRV/crotoxin was not toxic when i.v. inoculated in mice at a dose of crotoxin as high as 91 times its L.D50 or when s.c. inoculated at 42 times its LD50. On the other hand, crotoxin released from DRV/crotoxin retained its original toxicity. REV/crotoxin was found to be at least 1.9 times more toxic than DRV/crotoxin. The fact that DRV/crotoxin retained crotoxin more efficiently than REV/crotoxin may account for the difference in acute toxicity between the two preparations. DRV/crotoxin, when s.c. inoculated in mice, induced anti-crotoxin antibodies that protected animals against the lethal effect of Cdt venom. Following immunization with three doses of DRV/crotoxin (3 x 20 micrograms of crotoxin/mouse) and challenge with 8 x LD50 of Cdt venom, 75% of mice were protected. The DRV/crotoxin preparation was compared to crotoxin emulsified in Freund's adjuvant (FCA/crotoxin). DRV/crotoxin was found to be less toxic than FCA/crotoxin, and to induce lower levels of anti-crotoxin antibodies but similar levels of protection when inoculated at high doses (20 or 70 micrograms crotoxin/mouse). When DRV/crotoxin was adsorbed to alum at the time of immunization, it induced antibody and protection levels comparable to those produced by FCA/crotoxin.

Liposomes: from biophysics to the design of peptide vaccines.

Liposomes (lipid-based vesicles) have been widely studied as drug delivery systems due to their relative safety, their structural versatility concerning size, composition and bilayer fluidity, and their ability to incorporate almost any molecule regardless of its structure. Liposomes are successful in inducing potent in vivo immunity to incorporated antigens and are now being employed in numerous immunization procedures. This is a brief overview of the structural, biophysical and pharmacological properties of liposomes and of the current strategies in the design of liposomes as vaccine delivery systems.

Novel methods for the encapsulation of meglumine antimoniate into liposomes.

The antimonial drug, meglumine antimoniate, was successfully encapsulated in dehydration-rehydration vesicles and in freeze-dried empty liposomes (FDELs). High encapsulation efficiencies (from 28 to 58%) and low weight ratios of lipids to encapsulated antimony (from 1:0.15 to 1:0.3) were achieved. These formulations, contrary to those obtained by conventional methods, can be stored as intermediate lyophilized forms and reconstituted just before use. The efficacy of FDEL-encapsulated meglumine antimoniate was evaluated in hamsters experimentally infected with Leishmania chagasi. A significant reduction of liver parasite burdens was observed in animals treated with this preparation, when compared to control animals treated with empty liposomes. In contrast, free meglumine antimoniate was found to be inefficient when administered at a comparable dose of antimony. This novel liposome-based meglumine antimoniate formulation appears to be promising as a pharmaceutical product for the treatment of visceral leishmaniasis.

Distribution of liposome-encapsulated antimony in dogs.

The achievement of complete cure in dogs with visceral leishmaniasis is currently a great challenge, since dogs are the main reservoir for the transmission of visceral leishmaniasis to humans and they respond poorly to conventional treatment with pentavalent antimonials. In order to improve the efficacy of treatment, we developed a novel formulation for meglumine antimoniate based on the encapsulation of this drug in freeze-dried liposomes (LMA). The aim of the present study was to evaluate the biodistribution of antimony (Sb) in dogs following a single intravenous bolus injection of LMA. Four healthy male mongrel dogs received LMA at 3.8 mg Sb/kg body weight and were sacrificed 3, 48 and 96 h and 7 days later. Antimony was determined in the blood, liver, spleen and bone marrow. In the bone marrow, the highest Sb concentration was observed at 3 h (2.8 microg/g wet weight) whereas in the liver and spleen it was demonstrated at 48 h (43.6 and 102.4 microg/g, respectively). In these organs, Sb concentrations decreased gradually and reached levels of 19.1 microg/g (liver), 28.1 microg/g (spleen) and 0.2 microg/g (bone marrow) after 7 days. Our data suggest that the critical organ for the treatment with LMA could be the bone marrow, since it has low Sb levels and, presumably, high rates of Sb elimination. A multiple dose treatment with LMA seems to be necessary for complete elimination of parasites from bone marrow in dogs with visceral leishmaniasis.

Evaluation of the schistosomicidal efficacy of liposome-entrapped oxamniquine.

Oxamniquine (OXA) was successfully encapsulated in small unilamellar vesicles using a pH gradient method. This procedure led to a high drug encapsulation efficiency (> 85%) at a drug to lipid molar ratio of 1/10. Moreover, these liposomes were found to retain encapsulated OXA efficiently under dialysis conditions at 37 degrees C. Liposome-entrapped OXA (LOXA), OXA, and empty liposomes were tested against Schistosoma mansoni in a murine model. LOXA produced a significant reduction of the worm burden compared to the other preparations, when inoculated by subcutaneous route (s.c.) with 10 mg OXA/kg animal one day before the infection, and 3, 7, and 14 days after. However, LOXA was not effective when given 7 days before, or 35 days after infections. OXA, in the free form, was effective in relation to the untreated group, only when administered 3 days after the infection. Maximum effect of LOXA, with 97% reduction of the parasite number, was observed when the preparation was given s.c. one day before the infection. On the other hand, LOXA inoculated intraperitoneally one day before the infection didn't show any reduction of the parasite count. It can be concluded that LOXA is more effective than OXA for the treatment of experimental schistosomiasis, particularly when administered subcutaneously at a time close to the infection.

Protection against the toxic effects of Loxosceles intermedia spider venom elicited by mimotope peptides.

The venom of Loxosceles intermedia (Li) spiders is responsible for cutaneous lesions and other clinical manifestations. We previously reported that the monoclonal antibody LimAb7 can neutralize the dermonecrotic activity of crude Li venom. In this study, we observed that this antibody recognizes several proteins from the venom dermonecrotic fraction (DNF), including LiD1. Identifying the epitope of such a neutralizing antibody could help designing immunogens for producing therapeutic sera or vaccination approaches. To this aim, two sets of 25- and 15-mer overlapping peptides that cover the complete amino acid sequence of LiD1 were synthesized using the SPOT technique. None of them was recognized by LimAb7, suggesting that the epitope is discontinuous. Then, the screening of four peptide phage-display libraries yielded four possible epitope mimics that, however, did not show any obvious similarity with the LiD1 sequence. These mimotopes, together with a 3D model of LiD1, were used to predict with the MIMOP bioinformatic tool the putative epitope region (residues C197, Y224, W225, T226, D228, K229, R230, T232 and Y248 of LiD1) recognized by LimAb7. This analysis and the results of alanine-scanning experiments highlighted a few residues (such as W225 and D228) that are found in the active site of different SMases D and that may be important for LiD1 enzymatic activity. Finally, the only mimotope NCNKNDHLFACW that interacts with LimAb7 by SPOT and its analog NSNKNDHLFASW were used as immunogens in rabbits. The resulting antibodies could neutralize some of the biological effects induced by crude Li venom, demonstrating a mimotope-induced protection against L. intermedia venom.