Critical attributes of formulation and of elaboration process of PLGA-protein microparticles.

Low drug loading, burst effect during release and drug inactivation account for the main drawbacks of protein microencapsulation in poly(d,l-lactic-co-glycolic) acid (PLGA) matrix by the water-in oil-in water (W/O/W) solvent evaporation method. Thus, the current study was set to invest the critical attributes of formulation and of elaboration process which determine protein loading into microparticles as well as its further release, using albumin as protein model. NaCl concentration in the external aqueous phase, poly(vinyl alcohol) (PVA) concentration and mostly viscosity of both the internal aqueous phase and the organic phase were critical attributes for improving drug loading, with polymer molecular weight and hydrophobicity likewise directly related to albumin loading. In such a way, when using 0.5% PVA as internal aqueous phase the highest albumin loading was achieved. Optimized microparticles exhibited a sustained in vitro release of albumin over 130 days. The influence of the microencapsulation process on albumin stability and biological activity was evaluated by carrying out cell proliferation assays on PC12 cells with albumin released from microparticles. Such assay demonstrated that the microencapsulation procedure optimized in this study did not affect the biological stability of the microencapsulated protein.

Overcoming glucocorticoid resistances and improving antitumor therapies: lipid and polymers carriers.

PURPOSE: To improve chemotherapy protocols of lymphoid malignancies, by using polymeric and lipid microparticles as controlled delivery systems of dexamethasone, part of all combined chemotherapy protocols for its strong-inducing effect on malignant lymphoblasts. METHODS: Polymeric microparticles were prepared by the oil-in-water-emulsion cosolvent evaporation method, andlipid microparticles by spray drying. Their cytotoxic effects on GC-sensitive PC12 cells and GC-resistant PC3 cells were characterized by cell proliferation and apoptosis assays. RESULTS: Both elaboration methods rendered optimal-sized microparticles for parenteral administration with high drug loading. In vitro assays showed sustained dexamethasone release from polymeric microparticles over a month, whereas 100% dexamethasone release from lipid microparticles was achieved within 24 h. Similar PC12 cell death to that obtained with dexamethasone solution administered every 48 h was achieved with dexamethasone polymeric microparticles in 26-days assays. Dexamethasone solution and loaded polymeric microparticles induced apoptosis around 15.8 and 19.9%, respectively, after 2 days of incubation. Lipid microparticles increased further apoptosis induction in PC12 cells and, unlike dexamethasone solution and polymeric microparticles, showed antiproliferative effects on PC3 cells. CONCLUSIONS: Dexamethasone polymeric microparticles constitute an alternative to current dexamethasone administration systems in combined chemotherapy, whereas dexamethasone lipid microparticles represent a potential tool to revert glucocorticoid resistance.

New insights on culture and calcium signalling in neurons and astrocytes from epileptic patients.

Primary brain cell cultures are a useful tool for understanding the physiopathology of epilepsy and for searching new potential antiepileptic drugs. These cell types are usually prepared from murine species and few human models have been described. The main goal of this study is the establishment of experimental conditions to isolate and culture neurons and astrocytes from human brain and to test its functionality. The tissues came from antiepileptic drug-resistant epileptic patients undergoing surgery. Human neurons and astrocytes were isolated following an enzymatic and mechanical dissociation protocol. Cultures were viable for 3-6 weeks. Cytological characterization was performed by immunocytochemistry using specific antibodies against both neuron (anti-NeuN) and astrocyte (anti-GFAP) protein markers. In order to test their viability and functionality, cells were loaded with the fluorescent calcium probe fura-2 and variations in cytosolic calcium concentrations ([Ca2+]c) were measured by cell imaging. [Ca2+]c increases were evoked upon cell stimulation with high K+ (KCl 75 mM), glutamate (500 µM) or bicuculline (100 µM). Interestingly, spontaneous [Ca2+]c transients were also observed in some neuron-like cells. A novel unreported finding in this study has been the incorporation of human serum that was critical for cell functionality. The setting of these human cultures open the opportunity to new insights on culture and calcium signalling studies on the mechanism(s) of cell resistance to antiepileptic drugs, as well as to studies on plasticity, maturation and possible neurite emission for graft studies.

Calcium entry through L-type calcium channels causes mitochondrial disruption and chromaffin cell death.

Sustained, mild K+ depolarization caused bovine chromaffin cell death through a Ca(2+)-dependent mechanism. During depolarization, Ca(2+) entered preferentially through L-channels to induce necrotic or apoptotic cell death, depending on the duration of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) signal, as proven by the following. (i) The L-type Ca(2+) channel activators Bay K 8644 and FPL64176, more than doubled the cytotoxic effects of 30 mm K+; (ii) the L-type Ca(2+) channel blocker nimodipine suppressed the cytotoxic effects of K+ alone or K+ plus FPL64176; (iii) the potentiation by FPL64176 of the K+ -evoked [Ca(2+)](c) elevation was totally suppressed by nimodipine. Cell exposure to K+ plus the L-type calcium channel agonist FPL64176 caused an initial peak rise followed by a sustained elevation of the [Ca(2+)](c) that, in turn, increased [Ca(2+)](m) and caused mitochondrial membrane depolarization. Cyclosporin A, a blocker of the mitochondrial transition pore, and superoxide dismutase prevented the apoptotic cell death induced by Ca(2+) overload through L-channels. These results suggest that Ca(2+) entry through L-channels causes both calcium overload and mitochondrial disruption that will lead to the release of mediators responsible for the activation of the apoptotic cascade and cell death. This predominant role of L-type Ca(2+) channels is not shared by other subtypes of high threshold voltage-dependent neuronal Ca(2+) channels (i.e. N, P/Q) expressed by bovine chromaffin cells.

Mechanisms of blockade by the novel migraine prophylactic agent, dotarizine, of various brain and peripheral vessel contractility.

The novel antimigraineur, dotarizine, inhibited 5-HT (5 hydroxytryptamine)-evoked contractions of rabbit vertebral, aorta, femoral and mesenteric arteries, with IC(50)s of 1.35, 1.40, 0.52 and 1.09 microM, respectively. Flunarizine had little effect on these contractions, while ketanserin was more potent (IC(50)s of 0.17 microM for vertebral, 0.22 microM for aorta, 0.05 microM for femoral and 0.03 microM for mesenteric arteries). At 10 microM, dotarizine caused 40% blockade of K(+)-evoked contractions of rabbit aorta, and 70% inhibition of 5-HT-evoked responses; these values were 30% and 20% for 10 microM flunarizine. Contractions of rabbit aorta elicited by noradrenaline, angiotensin II or prostaglandin F(2alpha) were not affected by 10 microM dotarizine or flunarizine. Ketanserin shifted to the right, in parallel, the concentration-response curves for 5-HT in rabbit aorta; however, dotarizine caused a non-competitive type of blockade, increasing the maximum 5-HT contraction at 30 nM and decreasing it at 3 and 30 microM. K(+)-evoked contractions of rabbit aorta were halved by 3 microM dotarizine in a voltage-independent manner; flunarizine caused a delayed-type, non-reversible post-drug blockade, and exhibited some voltage-dependence. Blockade by nifedipine was voltage-dependent and fully reversible. Ca(2+)-evoked contractions of depolarised bovine middle cerebral arteries were blocked by 1--3 microM dotarizine in a non-surmountable manner. Contraction of these vessels evoked by electrical stimulation was blocked 50% and 70% by 1 and 3 microM dotarizine, respectively. Dotarizine (1--3 microM) also inhibited to a similar extent the K(+)-evoked [(3)H]noradrenaline release from cultured rat sympathetic neurones. These data suggest that the mechanism of blockade by dotarizine of cerebral vessels contractility has three components: (i) presynaptic inhibition of noradrenaline release; (ii) blockade of postsynaptic vascular 5-HT receptors; (iii) blockade of Ca(2+)entry into the vascular smooth muscle cell cytosol. The compound does not affect the vascular receptors for noradrenaline, angiotensin II or prostaglandin F(2alpha).

The sea anemone toxin Bc2 induces continuous or transient exocytosis, in the presence of sustained levels of high cytosolic Ca2+ in chromaffin cells.

We have isolated and characterized a new excitatory toxin from the venom of the sea anemone Bunodosoma caissarum, named Bc2. We investigated the mechanism of action of the toxin on Ca(2+)-regulated exocytosis in single bovine adrenal chromaffin cells, monitoring simultaneously fura-2 fluorescence measurements and electrochemical recordings using a carbon fiber microelectrode. Bc2 induced quantal release of catecholamines in a calcium-dependent manner. This release was associated with a sustained rise in cytosolic Ca(2+) and displayed two different patterns of response: a continuous discharge of prolonged duration that changed to a transient burst as the toxin concentration (or incubation time) increased. Continuous secretion was dependent on the activity of native voltage-dependent Ca(2+) channels and showed a pattern similar to that of alpha-latrotoxin; however, its kinetics adjusted better to that of continuous cell depolarization with high K(+) concentration. In contrast, transient secretion was independent of Ca(2+) entry through native voltage-dependent Ca(2+) channels and showed inhibition of late vesicle fusion that was accompanied by "freezing" of F-actin disassembly. These new features make Bc2 a promising new tool for studying the machinery of neurotransmitter release.

Ba(2+)-induced chromaffin cell death: cytoprotection by Ca(2+) channel antagonists.

Exposure of bovine adrenal medullary chromaffin cells to Ba(2+) ions (in the absence of Ca(2+) ions) caused their death, measured as lactate dehydrogenase (LDH) release. The concentration of Ba(2+) required to damage the cells by about 65% ranged between 1 and 10 mM (no Ca(2+) added); the required exposure time was rather brief (15 min-4 h). The simultaneous presence of Ca(2+), Mg(2+) or Zn(2+) together with Ba(2+) (2 mM, 4 h) afforded cyprotection (60-80%). Individual selective blockers of Ca(2+) channel subtypes afforded no protection. However, combined nifedipine (3 microM) plus omega-conotoxin MVIIC (3 microM) offered full protection. Substantial protection was also seen with the "wide-spectrum" Ca(2+) channel blockers penfluridol (0.3 microM), lubeluzole (3 microM), dotarizine (3 microM), flunarizine (3 microM), and mibefradil (3 microM). This protection was due to blockade of Ba(2+) entry through Ca(2+) channels because dotarizine (10 microM) inhibited the increase in cytosolic [Ba(2+)] seen in fura-2-loaded chromaffin cells. Once Ba(2+) accumulated in the cytosol, it was not extruded by the Na(+)/Ca(2+) exchanger, as shown by the prolonged and sustained elevation of the fura-2 signal. This contrasts with the fast dissipation of the fura-2 signal generated by [Ca(2+)](i) elevation. Thus, Ba(2+) overload can cause cell death by mechanisms similar to those reported for Ca(2+) overload and might be used as a novel and convenient tool to search for new cytoprotective compounds.

Voltage inactivation of Ca2+ entry and secretion associated with N- and P/Q-type but not L-type Ca2+ channels of bovine chromaffin cells.

1. In this study we pose the question of why the bovine adrenal medullary chromaffin cell needs various subtypes (L, N, P, Q) of the neuronal high-voltage activated Ca2+ channels to control a given physiological function, i.e. the exocytotic release of catecholamines. One plausible hypothesis is that Ca2+ channel subtypes undergo different patterns of inactivation during cell depolarization. 2. The net Ca2+ uptake (measured using 45Ca2+) into hyperpolarized cells (bathed in a nominally Ca2+-free solution containing 1.2 mM K+) after application of a Ca2+ pulse (5 s exposure to 100 mM K+ and 2 mM Ca2+), amounted to 0.65 +/- 0.02 fmol cell-1; in depolarized cells (bathed in nominally Ca2+-free solution containing 100 mM K+) the net Ca2+ uptake was 0.16 +/- 0.01 fmol cell-1. 3. This was paralleled by a dramatic reduction of the increase in the cytosolic Ca2+ concentration, [Ca2+]i, caused by Ca2+ pulses applied to fura-2-loaded single cells, from 1181 +/- 104 nM in hyperpolarized cells to 115 +/- 9 nM in depolarized cells. 4. A similar decrease was observed when studying catecholamine release. Secretion was decreased when K+ concentration was increased from 1.2 to 100 mM; the Ca2+ pulse caused, when comparing the extreme conditions, the secretion of 807 +/- 35 nA of catecholamines in hyperpolarized cells and 220 +/- 19 nA in depolarized cells. 5. The inactivation by depolarization of Ca2+ entry and secretion occluded the blocking effects of combined omega-conotoxin GVIA (1 microM) and omega-agatoxin IVA (2 microM), thus suggesting that depolarization caused a selective inactivation of the N- and P/Q-type Ca2+ channels. 6. This was strengthened by two additional findings: (i) nifedipine (3 microM), an L-type Ca2+ channel blocker, suppressed the fraction of Ca2+ entry (24 %) and secretion (27 %) left unblocked by depolarization; (ii) FPL64176 (3 microM), an L-type Ca2+ channel 'activator', dramatically enhanced the entry of Ca2+ and the secretory response in depolarized cells. 7. In voltage-clamped cells, switching the holding potential from -80 to -40 mV promoted the loss of 80 % of the whole-cell inward Ca2+ channel current carried by 10 mM Ba2+ (IBa). The residual current was blocked by 80 % upon addition of 3 microM nifedipine and dramatically enhanced by 3 microM FPL64176. 8. Thus, it seems that the N- and P/Q-subtypes of calcium channels are more prone to inactivation at depolarizing voltages than the L-subtype. We propose that this different inactivation might occur physiologically during different patterns of action potential firing, triggered by endogenously released acetylcholine under various stressful conditions.

Effects of dotarizine and flunarizine on chromaffin cell viability and cytosolic Ca2+.

Dotarizine (a novel piperazine derivative with antimigraine properties) and flunarizine (a Ca2+ channel antagonist) were compared concerning: first, their ability to cause chromaffin cell damage in vitro; second, the possible correlation of their octanol/water partition coefficients and those of another 28 compounds (i.e., Ca2+ channel antagonists, blockers of histamine H1 receptors, antimycotics, beta-adrenoceptor antagonists, neuroleptics), with their ability to cause cell damage; third, their capacity to protect the cells against the damaging effects of veratridine; and fourth, their capabilities to enhance the basal cytosolic Ca2+ concentration in fura-2-loaded single chromaffin cells, or to modify the pattern of [Ca2+]i oscillations elicited by veratridine. After 24-h exposure to 1-30 microM dotarizine, the viability of bovine adrenal chromaffin cells (measured under phase contrast or as lactate dehydrogenase, released into the medium) was similar to that of control, untreated cells; at 100 microM, 80% lactate dehydrogenase release was produced. At 1-3 microM flunarizine caused no cell damage; however 10 microM caused 20% lactate dehydrogenase release and 30 and 100 microM over 90% lactate dehydrogenase release. The time course of cell damage was considerably faster for flunarizine, in comparison to dotarizine. Out of 30 molecules tested (at 10 microM), having different octanol/water partition coefficients (log P), dotarizine was among the molecules causing no cell damage; flunarizine caused 20% cell loss, lidoflazine and verapamil over 50% cell loss, and penfluridol, draflazine, astemizole or nifedipine over 80% cell loss. No correlation was found between log P and cytotoxicity. Both dotarizine (10-30 microM) and flunarizine (3-10 microM) provided protection against veratridine-induced cell death; however, at 30 microM dotarizine afforded a pronounced protection while flunarizine enhanced the cytotoxic effects of veratridine. Dotarizine (30 microM) (but not flunarizine) caused a prompt transient elevation of the basal [Ca2+]i. Both compounds abolished the K+-induced increases of [Ca2+]i as well as the oscillations of [Ca2+]i induced by veratridine. The blocking effects of dotarizine were readily reversed after washout, while those of flunarizine were long-lasting. These differences might be relevant to the clinical use of dotarizine as an antimigraine drug.

Effects of the neuroprotectant lubeluzole on the cytotoxic actions of veratridine, barium, ouabain and 6-hydroxydopamine in chromaffin cells.

1. Incubation of bovine adrenal chromaffin cells with veratridine (10-100 microM) during 24 h, caused a concentration-dependent release of the cytosolic lactate dehydrogenase (LDH) into the bathing medium, an indicator of cell death. Lubeluzole or its R(-) enantiomer, R91154, did not enhance LDH release. Both lubeluzole and R91154 (0.3-10 microM) decreased the veratridine-induced LDH release. 2. Penfluridol did not increase LDH release at concentrations 0.003-1 microM; 3-10 microM increased LDH release to 50-60%, after 24 h exposure. Penfluridol (0.03-0.3 microM) did not protect against the cytotoxic effects of veratridine; at 1 microM, 15% protection was produced. Higher concentrations (3-10 microM) enhanced the cytotoxic effects of veratridine. 3. Ba2+ ions caused a concentration-dependent increase of LDH release. This cytotoxic effect was partially prevented by 3 microM lubeluzole and fully counteracted by 1 microM penfluridol. R91154 was less potent than lubeluzole and only protected against the lesion induced by 0.5 mM Ba2+. 4. Ouabain (10 microM during 24 h) increased LDH release to about 30%. Both lubeluzole (0.3-10 microM) and the lower concentrations of penfluridol (0.003-0.3 microM) prevented the ouabain cytotoxic effects. At higher concentrations (3 microM), penfluridol increased drastically the ouabain cytotoxic effects. 5. 6-Hydroxydopamine (6-OHDA) caused significant cytotoxic effects at 30 and 100 microM. Lubeluzole (3-10 microM) or penfluridol (0.03-0.3 microM) had no cytoprotective effects against 6-OHDA. 6. Lubeluzole (3 microM), R91154 (3 microM) and penfluridol (1 microM) blocked the current through Na+ channels in voltage-clamped chromaffin cells (I(Na)) by around 20-30%. Ca2+ current through Ca2+ channels (I(Ca)) was inhibited 57% by lubeluzole and R91154 and 50% by penfluridol. The effects of penfluridol were not washed out, but those of lubeluzole and R91154 were readily reversible. 7. Lubeluzole (3 microM) induced reversible blockade of the oscillations of the cytosolic Ca2+, [Ca2+]i, in fura-2-loaded cells exposed to 30 or 100 microM veratridine. Penfluridol (1 microM) inhibited those oscillations in an irreversible manner. 8. The results suggest that lubeluzole and its R-isomer caused cytoprotection against veratridine cell damage, by blocking the veratridine stimulated Na+ and Ca2+ entry, as well as the [Ca2+]i oscillations. The Ba2+ and ouabain cytotoxic effects were prevented more efficiently by penfluridol, likely by blocking the plasmalemmal Na+/Ca2+ exchanger. It remains dubious whether these findings are relevant to the reported neuroprotective action of lubeluzole in stroke; the doubt rests in the stereoselective protecting effects of lubeluzole in in vivo stroke models, as opposed to its lack of stereoselectivity in the in vitro model reported here.

Synergism between toxin-gamma from Brazilian scorpion Tityus serrulatus and veratridine in chromaffin cells.

Toxin-gamma (Tgamma) from the Brazilian scorpion Tityus serrulatus venom caused a concentration- and time-dependent increase in the release of norepinephrine and epinephrine from bovine adrenal medullary chromaffin cells. Tgamma was approximately 200-fold more potent than veratridine judged from EC50 values, although the maximal secretory efficacy of veratridine was 10-fold greater than that of Tgamma (1.2 vs. 12 microg/ml of catecholamine release). The combination of both toxins produced a synergistic effect that was particularly drastic at 5 mM extracellular Ca2+ concentration ([Ca2+]o), when 30 microM veratridine plus 0.45 microM Tgamma were used. Tgamma (0.45 microM) doubled the basal uptake of 45Ca2+, whereas veratridine (100 microM) tripled it. Again, a drastic synergism in enhancing Ca2+ entry was seen when Tgamma and veratridine were combined; this was particularly pronounced at 5 mM [Ca2+]o. Veratridine induced oscillations of cytosolic Ca2+ concentration ([Ca2+]i) in single fura 2-loaded cells without elevation of basal levels. In contrast, Tgamma elevated basal [Ca2+]i levels, causing only small oscillations. When added together, Tgamma and veratridine elevated the basal levels of [Ca2+]i without causing large oscillations. Tgamma shifted the current-voltage (I-V) curve for Na+ channel current to the left. The combination of Tgamma with veratridine increased the shift of the I-V curve to the left, resulting in a greater recruitment of Na+ channels at more hyperpolarizing potentials. This led to enhanced and more rapid accumulation of Na+ in the cell, causing cell depolarization, the opening of voltage-dependent Ca2+ channels, and Ca2+ entry and secretion.