Current management of breakthrough cancer pain according to physicians from pain units in Spain.

PURPOSE: Current evidence suggests the need to improve the management of breakthrough cancer pain (BTcP). For this reason, we aimed to assess the opinion of a panel of experts composed exclusively of physicians from pain units, who play a major role in BTcP diagnosis and treatment, regarding the key aspects of BTcP management. METHODS: An ad hoc questionnaire was developed to collect real-world data on the management of BTcP. The questionnaire had 5 parts: (a) organizational aspects of pain units (n = 12), (b) definition and diagnosis (n = 3), (c) screening (n = 3), (d) treatment (n = 8), and (e) follow-up (n = 7). RESULTS: A total of 89 pain-unit physicians from 13 different Spanish regions were polled. Most of them agreed on the traditional definition of BTcP (78.9%) and the key features of BTcP (92.1%). However, only 30.3% of participants used the Davies' algorithm for BTcP diagnosis. Respondents preferred to prescribe rapid-onset opioids [mean 77.0% (SD 26.7%)], and most recommended transmucosal fentanyl formulations as the first option for BTcP. There was also considerable agreement (77.5%) on the need for early follow-up (48-72 h) after treatment initiation. Finally, 65.2% of participants believed that more than 10% of their patients underused rapid-onset opioids. CONCLUSIONS: There was broad agreement among pain experts on many important areas of BTcP management, except for the diagnostic method. Pain-unit physicians suggest that rapid-onset opioids may be underused by BTcP patients in Spain, an important issue that need to be evaluated in future studies.

Photosensitization of skin fibroblasts and HeLa cells by three chlorin derivatives: Role of chemical structure and delivery vehicle.

The chemical nature of the sensitizer and its selective uptake by malignant cells are decisive to choose an appropriate biocompatible carrier, able to preserve the photosensitizing characteristics of the dye. In this paper we demonstrate the photodynamic properties of three chlorins, derived from chlorophyll a, and the usefulness of liposomal carriers to design pharmaceutical formulations. The chlorins have been quantitatively incorporated into stable liposomes obtained from a mixture of L-alpha-palmitoyloleoylphosphatidylcholine and L-alpha-dioleoylphosphatidylserine in a 13.5:1.5 molar ratio (POPC/OOPS-liposomes). The chlorin uptake by skin fibroblasts increases steadily, reaching in all cases a plateau level dependent on both the chlorin structure and the vehicle employed. The photophysical properties of the three chlorins in THF are nearly identical and fulfill the requirements for a PDT photosensitizer. Incorporation of chlorins into liposomes induces important changes in their photophysics, but does not impair their cellular uptake or their cell photosensitization ability. In fact we observe in the cells the same photophysical behavior as in THF solution. Specifically, we demonstrate, by recording the near-IR phosphorescence of 1O2, that the chlorins are able to photosensitize the production of 1O2 in the cell membrane. The cell-photosensitization efficiency depended on the chlorin and cell line nature, the carrier, and the length of pre-incubation and post-irradiation periods. The high photodynamic activity of chlorin-loaded liposomes and the possibility to design liposomal carriers to achieve a specific target site favors this approach to obtain an eventual pharmaceutical formulation.

Incorporation of hydrophobic porphyrins into liposomes: characterization and structural requirements.

The ability of photosensitisers to give reactive oxygenated products is considered decisive for photodynamic applications, but the hydrophobic nature of many porphyrins makes necessary to obtain suitable pharmaceutical formulations. This paper reports the structural photosensitiser features that allow the preparation of stable liposomal formulations. Metallated and non-metallated TPPs and TPyPs and different lipid/porphyrin ratios were considered in order to procure liposomal preparations containing porphyrin concentrations adequate to necessary doses. The results show that the incorporation of porphyrins into liposomes can be related with their ability to form aggregates in a watery media. Thus, ZnTPP, which structural properties avoid the formation of aggregates, was efficiently incorporated into stable liposomes. Moreover, the efficient generation of singlet oxygen by ZnTPP liposomal suspensions has been shown. Because of this, the synthesis of hydrophobic porphyrin derived structures or other sensitisers, which do not aggregate in a watery media and with Q-bands shifted to higher lambda values than ZnTPP, will be efficiently incorporated into liposomes and useful for clinical applications.

In vitro cytotoxic study of immunoliposomal doxorubicin targeted to human CD34(+) leukemic cells.

The expression of CD34 antigen in acute myelogenous leukemias is considered an unfavourable prognosis marker for response to anticancer drugs and duration of remission. This study investigated the applicability of long-circulating immunoliposomes loaded with doxorubicin targeted to CD34 antigen present on MDR(+) human myelogenous leukemia KG-1a cell line. Immunoliposomal doxorubicin showed a higher cytotoxicity against KG-1a cells than non-targeted liposomal doxorubicin, but it did not improve over that of free drug. Although no reversal of doxorubicin resistance was found to occur through its liposomal encapsulation, a therapeutic benefit can be obtained by the selective cytotoxicity observed. Endocytosis studies demonstrated that, after binding to CD34 antigen, the immunoliposomes are not internalized by the KG-1a cells and so the cytotoxic effect might be due to drug released into the space near the cell membrane. Thus, immunotargeting of liposomal doxorubicin to CD34(+) leukemic cells may only provide an ex vivo strategy for site-selective CD34(+) leukemia cell killing.

Interaction of tocopherols and phenolic compounds with membrane lipid components: evaluation of their antioxidant activity in a liposomal model system.

This paper describes the use of complex liposomes as real membrane models to evaluate the potential benefits of several antioxidants in relation to lipid peroxidation. The xanthine oxidase/Fe(3+)-ADP-EDTA and the Fe(2+)/H2O2 systems have been used to generate hydroxyl radicals and the water soluble azo-compound 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) to generate carbon centered radicals (A*) by thermal decomposition. The antioxidant behavior of the rosemary and citrus plant extracts and vitamin-E and vitamin-E acetate alpha-tocopherols have been analyzed. The order of effectiveness in avoiding radical chain reactions has been established by using the colorimetric thiobarbituric acid reaction and the fluorescent probe DPH-PA. ESR spectroscopy has been used to carry out the pursuit of the oxidation processes on the basis of the identification of the radical species resulting from the oxidant system and the ability of the antioxidants to act as scavengers for hydroxyl and AAPH-derived radicals. The modification of the main transition temperature for the lipid mixture and the splitting of the calorimetric peak in the presence of the antioxidants were demonstrated by differential scanning calorimetry. The results obtained showed that the phenols-containing plant extracts and alpha-tocopherols perturb the phase behavior of the BBE lipid bilayer and have a fluidifying effect that could favor the known antioxidant capability and scavenging characteristics of these compounds. 31P-NMR results could be interpreted as, after the incorporation of these antioxidants, those lipid molecules interacting with antioxidants give rise to lamellar phase spectral components with resonance position at lower fields or to isotropic signals in accordance with a higher motion of their phosphate groups.

Antioxidant and pro-oxidant effect of the thiolic compounds N-acetyl-L-cysteine and glutathione against free radical-induced lipid peroxidation.

The antioxidant ability of thiol compounds has been the subject of much of the current research about oxidative stress. The direct scavenging of hydroxyl radicals by thiols has been suggested as their protection mechanisms. Nevertheless, the interaction of thiols with reactive radicals can generate thiyl radicals, which, in turn, may impart a pro-oxidant function. The purpose of this study has been to establish the effect of the thiol compounds N-acetyl-L-cysteine (NAC) and glutathione (GSH) against the peroxidative processes involving membrane lipids. The results obtained support the ability of NAC and GSH to suppress the 2,2'-azobis-(2-amidinopropane) dihydrochloride (AAPH)-dependent or to enhance the Fe2+/H2O2-dependent oxidative actions. The evaluation of thiobarbituric acid reactive substances (TBARS) production, the study of the influence of oxidants on membrane fluidity and the measurements of the changes in the fluorescence of bilayer probes, such as 3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid (DPH-PA), have shown the antioxidant and pro-oxidant effects of both NAC and GSH. Also their dependence on the nature of the radicals generated by the oxidative systems used has been shown. The use of ESR spectroscopy has allowed us to establish the ability of these compounds to scavenge the AAPH-derived radicals, to determine the formation of thiyl radicals in the iron-mediated oxidation and to evaluate the enhanced production of hydroxyl radicals by NAC and GSH.

Preparation of long-circulating immunoliposomes using PEG-cholesterol conjugates: effect of the spacer arm between PEG and cholesterol on liposomal characteristics.

Poly(ethylene glycol)-coated liposomes were prepared with two new synthesised pegylated cholesterol (Chol) derivatives linked via carbamate bond. Poly(ethylene glycol) (PEG) was directly linked to Chol (PEG-Chol) or through a space arm of diaminebutane (PEG-L-Chol). In buffer, the physicochemical properties of PC/Chol liposomes (2/1, molar ratio) containing up to 10 mol% of pegylated Chol derivatives did not change significantly and the PEG layer at liposome surface inhibited the agglutination of biotin-liposomes induced by streptavidin. On the other hand, in serum, PEG-L-Chol seemed to reduce the interactions of liposomes with serum proteins, much more than PEG-Chol. The low steric hindrance of PEG-Chol derivative may be due to the slow conformational transition rate of the polymer, since PEG may be deeper located in the membrane. The coupling efficiency of the ligand to the functionalised amino group at the polymer end was also affected, but, its antigen-binding activity was preserved. The basic physical-chemical characteristics studied in this work are relevant to assess the application of pegylated Chol liposomes as drug delivery systems.

Preparation of PEG-grafted immunomagnetoliposomes entrapping citrate stabilized magnetite particles and their application in CD34+ cell sorting.

Immunomagnetic systems have been used for positive selection of a cell fraction from a mixture using appropriate surface markers with satisfactory results, as haematopoietic CD34+ cells. This work reports on the development of poly(ethylene glycol)-grafted (PEG) immunoliposomes loaded with citrate-magnetite stabilized particles as the separation vehicles for immunomagnetic separations. The magnetic ferrofluid was encapsulated into PEG-liposomes by the DRV methodology. The magnetoliposomes had a liposomal size of approximately 450 nm and a Fe/lipid molar ratio of 1.52+/-0.26, and were retained in the magnetic field created by the MiniMACS system. Anti-CD34 immunomagnetoliposomes with 100 mAb/vesicle were prepared by coupling the My10 mAb and bound specifically for CD34+ KG-1a cells in culture and in mixtures with CD34-cells (CHO or Jurkat). The magnetic cell sorting was carried out in cell mixtures KG-1a/CHO or KG-1a/Jurkat with different initial% of CD34+ Kg-1a cells. For 10(6) positive cells and 100 microM of immunomagnetoliposomes, the capture efficiency was > 85% and independent of the starting percentage of CD34+ cells. The decrease of the final purity, when the starting percentage of CD34+ cells decreases and, dependent of the CD34- cell line used, point to the degree of non-specific cell binding of My10-immunomagnetoliposomes as being crucial, among of the methodological aspects as the number of starting CD34+ cells. The CD34+ cells isolated retained the viability with an estimated recovery of 45-50%.

Preparation of immunoliposomes bearing poly(ethylene glycol)-coupled monoclonal antibody linked via a cleavable disulfide bond for ex vivo applications.

Several methods for the preparation of sterically stabilized immunoliposomes (SIL) have recently been described. This report examines an established method for coupling anti-CD34 My10 mAb to poly(ethylene glycol)-liposomes (PEG-liposomes) containing the anchor pyridyldithiopropionylamino-PEG-phosphatidylethanolamine (PDP-PEG-PE) via a cleavable disulfide bond. Efficient attachment of pyridyldithio-derivatized mAb took place (equivalent to coupling ca. 70% of total input protein) at 2 mol percent of the functionalized PEG-lipid. The My10-SIL bound specifically to CD34+ cells (human leukemic KG-1a and hematopoietic progenitor cells) and the extent of binding was a function of liposomal lipid concentration, the mAb density in the liposome surface and the CD34 cell expression. In mixtures with CD34- cells (CHO or Jurkat), CD34+KG-1a cells were determined by flow cytometry at percentages (1-4%) similar to those reported in clinical samples (such as cord blood, mobilized peripheral blood and bone marrow) using a direct immunostaining with My10-SIL. The disulfide bond was stable in cell culture medium (10% of fetal calf serum) during 8 h and cell-bound SIL can be released from cells by treatment with dithiothreitol as reducing agent under mild conditions (1 h of incubation with 50 mM DTT at 20 degrees C). SIL binding and subsequent dithiothreitol treatment did not influence the cell viability. Our approach should contribute to the development of targetable liposomal vehicles to CD34+ cells for use in ex vivo conditions as sorting of hematopoietic stem cells.

Aggregation and fusion of vesicles composed of N-palmitoyl derivatives of membrane phospholipids.

N-Acylphosphatidylethanolamines and N-acylphosphatidylserines have been isolated from mammalian cells and have been associated with some tissue degenerative changes, although the relationship between their synthesis and the uncontrolled sequence of events that ends in irreversible tissue damage is not completely established. Our results show that monovalent and divalent cations induce aggregation and fusion of liposomes constituted by N-palmitoylphosphatidylethanolamine (NPPE) and N-palmitoylphosphatidylserine (NPPS). The effectiveness of cations to induce the aggregation of NPPE and NPPS liposomes is Ca2+ > Mg2+ >> Na+. NPPS liposomes aggregate at lower concentrations of divalent cations than NPPE liposomes, but with sodium NPPE liposomes aggregate to a higher extent than NPPS liposomes. The reaction order for the aggregation processes depends on the lipid and the cation nature and range from 1.04 to 1.64. Dynamic light scattering shows an irreversible increase of the size of the aggregates in the presence of all cations tested. The irreversibility of the aggregation process and the intermixing of bilayer lipids, as studied by resonance energy transfer assay, suggest that fusion, rather than aggregation, occurs. The existence of a real fusion was demonstrated by the coalescence of the aqueous contents of both NPPS and NPPE liposomes in the presence of either monovalent or divalent cations. The different binding sensitivity of Ca2+ to NPPS and NPPE liposomes, determined by zeta potential measurements, agrees with the results obtained in the aggregation and fusion assays. Our results suggest that the synthesis in vivo of N-acylated phospholipids can introduce important changes in membrane-mediated processes.

A novel strategy affords high-yield coupling of antibody to extremities of liposomal surface-grafted PEG chains.

Several methodologies for the preparation of polyethylene glycol-grafted immunoliposomes have been developed by attaching antibodies to the terminus of the polymer. Unilamellar liposomes were prepared containing a combination of a functionalized polyethylene glycol(3400) and an inert polyethylene glycol(2000) phosphatidylethanolamine derivate up to 5 mol%. The greater length of the functionalized polyethylene glycol derivate did not alter the liposomal sterical stability or the remote loading of doxorubicin. Anti-CD34 immunoliposomes were prepared by the reaction of maleimide-derivatized My10 antibody with generated thiol groups at the periphery of the liposomes and efficiencies of nearly 100% were obtained. The greater accessibility of the reactive group makes this strategy more efficient than others described. The immunoliposomes prepared bound specifically to CD34+ cells.

N-stearoyl-phosphatidylserine: synthesis and role in divalent-cation-induced aggregation and fusion.

N-Acylphosphatidylserines have been isolated from intact and injured tissues, but the participation of such acidic phospholipids in membrane aggregation and fusion has not been demonstrated. We have synthesized N-stearoylphosphatidylserine (NSPS) and examined divalent-cation-induced aggregation of NSPS-liposomes, which leads to membrane destabilization and fusion. The purified lipid was characterized by its chromatographic and spectroscopic (infrared and 1H nuclear magnetic resonance) properties and by its chemical degradation pattern. Aggregation of unilamellar NSPS-liposomes was studied as a function of calcium and magnesium concentration. The ability of calcium and magnesium to induce vesicle aggregation is higher for phosphatidylserine (PS)-liposomes (threshold concentration 1.5 mM for calcium and 4.6 mM for magnesium) than for NSPS-liposomes (threshold concentration 2.8 mM for calcium and 6.6 mM for magnesium). The irreversibility of the aggregation reactions after adding EDTA suggests that vesicle fusion might occur in the presence of calcium and magnesium. Preliminary studies, based on mixing of both lipid and internal aqueous contents, show that fusion rather than aggregation of NSPS-liposomes occurs in the presence of calcium ions. The tendency of NSPS-liposomes to aggregate at higher cation concentrations than PS-liposomes suggests that N-acylation of phosphatidylserine protects the membrane against degenerative damage caused by aggregation and fusion.

Preparation of immunoliposomes directed against CD34 antigen as target.

The My-10 monoclonal antibody has facilitated the search of haematopoietic stem cells by recognizing selectively the human CD34 antigen. In the present work, My-10 immunoliposomes directed specifically against CD34+ cells were prepared, characterized and tested in vitro. Binding to target cells at 4 degreesC of immunoliposomes containing carboxyfluorescein as aqueous marker was evaluated by flow cytometry and fluorescence microscopy. These immunoliposomes demonstrated their capacity to bind specifically to CD34+ cells. Studies have shown that 9 antibodies/vesicle were sufficient to obtain a good binding efficiency. The product was stable over one month at 4 degreesC in terms of leakage of encapsulated carboxyfluorescein, particle size and antigen binding capacity.

Synthesis of 1,2-diacyl-sn-glycerophosphatidylserine from egg phosphatidylcholine by phosphoramidite methodology.

A simple chemical method for the synthesis of 1,2-diacyl-sn-glycerophosphatidylserine (PS), with the same fatty acid composition in the sn-1 and sn-2 glycerol positions as egg phosphatidylcholine (PC), is described. PS synthesis was carried out by a phosphite-triester approach, using 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (phosphoramiditate) as the phosphorylating agent, for the formation of phosphate linkage between serine and diacylglycerol. 1,2-Diacylglycerol, obtained from PC hydrolysis by phospholipase C, was coupled with N-t-BOC-L-serinebenzhydryl ester phosphoramidite with tetrazole as catalyst. Phosphite-triester was oxidized to the corresponding phosphate-triester with 30% H2O2 in CH2Cl2. The cyanoethyl group was removed by addition of an Et3N/CH3 CN/pyridine mixture, and trifluoroacetic acid was used to eliminate the protecting groups of O-(1,2-diacylglycero-3-phospho)-N-t-BOC-serinebenzhydryl ester. Purified PS was identified by thin-layer chromatography, infrared, and 1H nuclear magnetic resonance.

N-palmitoylphosphatidylethanolamine stabilizes liposomes in the presence of human serum: effect of lipidic composition and system characterization.

Liposomes containing negatively-charged phospholipid, N-palmitoylphosphatidylethanolamine (NPPE) were examined for stability in the presence of human serum, using the release of the entrapped 5,6-carboxyfluorescein as an aqueous marker. Either small unilamellar vesicles (SUV) or large unilamellar vesicles (LUV) were used. Incorporation of NPPE into PC SUV decreases leakage in the presence of serum or phosphate-buffered saline, no strictly related to size increase observed and to the surface negative charge present. The stabilizing effect of NPPE and Chol were synergistic. Inhibition of destabilization induced by serum of PC/Chol liposomes was observed when NPPE concentrations were above 12 mol%. Change in the membrane fluidity or incorporation of a monosialoganglioside into liposomes do not significantly change the half-life of liposomes in the presence of a high NPPE concentration. Incorporation of NPPE into PC/Chol liposomes increases membrane rigidity which does not change after serum incubation. The presence of NPPE in liposomes decreases lipid transfer/exchange between liposomes and lipoproteins although the same amount of serum proteins were incorporated as in PC/Chol liposomes. As expected, these proteins are accessible to trypsin digestion. In accordance with these results, the liposome agglutination assay shows no steric barrier activity. As a whole, the results obtained in this paper suggest a complex mechanism for stabilization of NPPE containing liposomes in human serum.

Role of headgroup structure in the phase behaviour of N-acylethanolamine phospholipids: hydrogen-bonding ability and headgroup size.

The physical properties of aqueous dispersions of N-acylphosphatidylethanolamine from natural origin with long N-acyl chain (NAPE) and headgroup modified analogues have been studied. N-Acylation of PE causes a significant increase in the gel-to-liquid crystalline lamellar phase transition temperature in contrast with saturated N-acyl(dipalmitoyl) PEs, and in addition it does not restrict the headgroup rotational mobility in gel phase. The results agree with the increase of hydration of the phosphate group compared with that in PE and suggest the formation of hydrogen bonds between amide groups. The modifications introduced modulate the headgroup size and their hydrogen bonding capability. An increasing number of methylene groups between the phosphate and amide groups does not modify the phase behaviour observed. N-methylation of the amide group, which prevents the possibility of intermolecular hydrogen bond formation, decreases the melting temperature and the cooperativity of the phase transition and does not change the phase behaviour, while the hydration at the ester carbonyl groups level is decreased. On the other hand, the addition of N-ethyl substituent to the amide group or substitution of an ester group for this group increases its tendency to form structures with inverted geometries. The behaviour of these compounds suggests that hydration forces must be more important than considerations of the lipid dynamic shape in predicting the relative stabilities of lamellar vs. non-lamellar phases for NAPEs with long saturated N-acyl chain.

Incorporation of N-acylethanolamine phospholipids into egg phosphatidylcholine vesicles: characterization and permeability properties of the binary systems.

We have studied the effect of the N-acylphosphatidylethanolamine (N-acylPE) on the permeability properties of liposomes composed primarily of egg phosphatidylcholine using a fluorescent anionic dye, carboxyfluorescein, as model solute. Leakage from liposomes decreased and vesicle size increased with increasing N-acylPE content. In addition, measurement of the trapped aqueous space, using the same dye marker, showed a correlation between trapped volume and vesicle size determined by dynamic light scattering. Permeability parameters were calculated according to the pseudo-first-order analysis. It appears that N-acylPE stabilizes liposomes at least in part through its ability to impart surface negative charge, in accord with the results obtained with potassium chloride as encapsulated solute. These results agreed well with osmotic response of anionic lipid vesicles. Cholesterol stabilizes N-acylPE liposomes in a proportional manner to the molar fraction of the effector.

Cation-induced aggregation and fusion of N-acyl-N-methyl-phosphatidylethanolamine vesicles.

Aggregation and fusion of unilamellar vesicles consisting of N-acyl-N-methylphosphatidylethanolamine were studied as a function of mono- and divalent cation concentrations. The aggregation reactions were irreversible processes, as demonstrated by changes in monovalent ion concentrations and by the addition of ethylenediaminetetraacetic acid (EDTA) to chelate divalent cations, suggesting the possibility of some cation-induced vesicle fusion. An increase in the NaCl ionic strength of the vesicle suspension solutions diminishes the threshold concentration for Li+ and K+ and increases that corresponding to Mn2+, Mg2+ and Ca2+. However NaCl concentrations above 300 mM yield smaller threshold values for the divalent cation-induced processes, probably due to the increased size of phospholipid vesicles as the ionic strength of the medium increases.