Small-scale preparation of perfluorocarbon-nanoemulsions utilizing dual centrifugation.

BACKGROUND: Perfluorocarbon-nanoemulsions (PFC-NE) made of PFC and phospholipids (PL) by homogenization are optimal for in vivo-19F labelling of monocytes and subsequently of inflamed tissues in magnetic resonance imaging (MRI). Necessary requirements for in vivo use of PFC-NE are sterility, suitable droplet sizes and the absence of immune activating liposomes, which are a typical byproduct of the homogenization process. METHODS AND RESULTS: To meet these requirements, we developed an aseptic in-vial preparation technique for PFC-NE based on dual centrifugation (DC) by testing different PFC/phospholipid ratios as well as the application of additives. Two different PFC, perfluorooctylbromide (PFOB) and perfluoro-15-crown-5-ether (PFCE), were investigated. Particle sizes were assessed by dynamic light scattering and NE morphology by cryoTEM. DC homogenization was optimal when using an excess of PL (8.7 % (m/m) of utilized PFC, z-ave: 180 nm, pdi: 0.2). A purification approach by centrifugation was implemented to remove liposomes formed from the excess of PL during homogenization. The purification success was proven by phospholipid assay and PFC quantification via density and sound velocity measurements. CONCLUSION: DC in combination with a short centrifugation is a fast and reliable way of small-scale aseptic PFC-NE production for 19F MRI passive-targeting experiments of monocytes and inflamed tissues.

A new liposomal formulation of Gemcitabine is active in an orthotopic mouse model of pancreatic cancer accessible to bioluminescence imaging.

Despite its rapid enzymatic inactivation and therefore limited activity in vivo, Gemcitabine is the standard drug for pancreatic cancer treatment. To protect the drug, and achieve passive tumor targeting, we developed a liposomal formulation of Gemcitabine, GemLip (Ø: 36 nm: 47% entrapment). Its anti-tumoral activity was tested on MIA PaCa-2 cells growing orthotopically in nude mice. Bioluminescence measurement mediated by the stable integration of the luciferase gene was employed to randomize the mice, and monitor tumor growth. GemLip (4 and 8 mg/kg), Gemcitabine (240 mg/kg), and empty liposomes (equivalent to 8 mg/kg GemLip) were injected intravenously once weekly for 5 weeks. GemLip (8 mg/kg) stopped tumor growth, as measured via in vivo bioluminescence, reducing the primary tumor size by 68% (SD +/- 8%; p < 0.02), whereas Gemcitabine hardly affected tumor size (-7%; +/- 1.5%). In 80% of animals, luciferase activity in the liver indicated the presence of metastases. All treatments, including the empty liposomes, reduced the metastatic burden. Thus, GemLip shows promising antitumoral activity in this model. Surprisingly, empty liposomes attenuate the spread of metastases similar to Gemcitabine and GemLip. Further, luciferase marked tumor cells are a powerful tool to observe tumor growth in vivo, and to detect and quantify metastases.

Inhibition of 14-kDa PLA2 by 2-acylamino-alkylphospholipids: the influence of amide acidity.

2-Acylamino-alkyl phospholipids are potent competitive inhibitors of 14-kDa phospholipases A2 (e.g., human nonpancreatic secretory PLA2). As concluded from X-ray studies the amide hydrogen of these inhibitors forms a hydrogen bond to His-48 in the active site of the enzyme. We investigated the quantitative contribution of this hydrogen bond to inhibition using especially designed inhibitors that bear different acyl chains with and without electron withdrawing or donating substituents, thus differing in amide acidity. Relative free enthalpies DeltaDeltaG of enzyme-inhibitor complex formations were calculated from Xi(50) values determined by pH-stat titration using a mixed micelles assay and PLA2 from Naja mocambique mocambique. A quantitative relationship between amide acidity and DeltaDeltaG values is presented. Comparison of isoacidic and isosteric inhibitors reveals that (i) the hydrogen bond of the amide proton to His-48 is crucial for strong PLA2 inhibition, (ii) regardless of the headgroup unsubstituted N-acyl groups result in optimal amide acidity for PLA2 inhibition and (iii) the exceptionally strong inhibition by acetamides and the isosteric fluoroacetamides is due to an additional steric effect.

Biophysical and lipofection studies of DOTAP analogs.

In order to investigate the relationship between lipid structure and liposome-mediated gene transfer, we have studied biophysical parameters and transfection properties of monocationic DOTAP analogs, systematically modified in their non-polar hydrocarbon chains. Stability, size and (by means of anisotropy profiles) membrane fluidity of liposomes and lipoplexes were determined, and lipofection efficiency was tested in a luciferase reporter gene assay. DOTAP analogs were used as single components or combined with a helper lipid, either DOPE or cholesterol. Stability of liposomes was a precondition for formation of temporarily stable lipoplexes. Addition of DOPE or cholesterol improved liposome and lipoplex stability. Transfection efficiencies of lipoplexes based on pure DOTAP analogs could be correlated with stability data and membrane fluidity at transfection temperature. Inclusion of DOPE led to rather uniform transfection and anisotropy profiles, corresponding to lipoplex stability. Cholesterol-containing lipoplexes were generally stable, showing high transfection efficiency at low relative fluidity. Our results demonstrate that the efficiency of gene transfer mediated by monocationic lipids is greatly influenced by lipoplex biophysics due to lipid composition. The measurement of fluorescence anisotropy is an appropriate method to characterize membrane fluidity within a defined system of liposomes or lipoplexes and may be helpful to elucidate structure-activity relationships.

Targeting of endothelial KDR receptors with 3G2 immunoliposomes in vitro.

Immunoliposomes (IL) containing anti-angiogenic drugs directed selectively to the easily accessible kinase insert domain containing receptor (KDR) vascular endothelial growth factor (VEGF), which is predominantly expressed on tumour vessels are a promising tool to inhibit tumour angiogenesis. To explore this strategy, we have prepared fluorescent-labelled IL presenting antibodies against the KDR receptor (3G2) on their surface. 3G2-IL were composed of egg phosphatidylcholine and cholesterol (6:4), containing 2 mol% of the new thiol reactive linker lipid O-(3-cholesteryloxycarbonyl)propionyl-O'-m-maleimido-benzoyl tetraethylene glycol. Specific binding of 3G2-IL to immobilised recombinant KDR was used to show the maintenance of sufficient immunoreactivity of 3G2 antibodies upon the coupling procedure. 3G2-IL bound to Chinese hamster ovarian (CHO) cells stably transfected to overexpress KDR to a five times higher amount as compared to mock-transfected CHO cells. Subsequently, specific binding of 3G2-IL to KDR could also be demonstrated on KDR expressing cells, human umbilical vein endothelial cells and human microvascular endothelial cells, whereas only low binding of 3G2-IL to NIH-3T3 mouse fibroblast cells, which do not express KDR, was found. The binding of 3G2-IL to KDR receptors could not be blocked by VEGF, suggesting that the binding site for VEGF is not identical with the epitope recognised by 3G2. We could demonstrate that 3G2-IL is able to bind in vitro even in the presence of high levels of VEGF.

Alkylphosphocholine-induced glioma cell death is BCL-X(L)-sensitive, caspase-independent and characterized by massive cytoplasmic vacuole formation.

Alkylphosphocholines (APC) are candidate anticancer agents. We here report that APC induce the formation of large vacuoles and typical features of apoptosis in human glioma cell lines, but not in immortalized astrocytes. APC promote caspase activation, poly(ADP-ribose)-polymerase (PARP) processing and cytochrome c release from mitochondria. Adenoviral X-linked inhibitor of apoptosis (XIAP) gene transfer, or exposure to the caspase inhibitor, benzyloxycarbonyl-Val-Ala-DL-Asp-fluoro-methylketone zVAD-fmk, blocks caspase-7 and PARP processing, but not cell death, whereas BCL-X(L) blocks not only caspase-7 and PARP processing but also cell death. APC induce changes in Delta Psi m in sensitive glioma cells, but not in resistant astrocytes. The changes in Delta Psi m are unaffected by crm-A (cowpox serpin-cytokine response modifier protein A), XIAP or zVAD-fmk, but blocked by BCL-X(L), and are thus a strong predictor of cell death in response to APC. Free radicals are induced, but not responsible for cell death. APC thus induce a characteristic morphological, BCL-X(L)-sensitive, apparently caspase-independent cell death involving mitochondrial alterations selectively in neoplastic astrocytic cells.

Immuno-magnetoliposomes targeting activated platelets as a potentially human-compatible MRI contrast agent for targeting atherothrombosis.

To detect unstable atherosclerotic plaques early and noninvasively would be of great clinical interest. Activated platelets are an interesting molecular target for detecting early lesions or unstable plaques. We therefore developed an MRI contrast agent consisting of magnetoliposomes (ML) linked to an antibody (anti-LIBS) specifically targeting the ligand-induced binding site of the activated GPIIb/IIIa receptor of platelets. ML were prepared by dual centrifugation (DC). ML pegylation up to a total PEG content of 7.5 mol% positively influenced the stability and amount of entrapped SPIOs, and also reduced SPIO-membrane interactions, while higher PEG contents destabilized PEG-ML. Stable anti-LIBS-ML with high amounts of entrapped SPIOs (∼86%, ∼0.22 mol Fe/mol liposomal lipid) and high MRI sensitivity (relaxivity r2 = 422 s(-1) mM(-1) and r2(∗) = 452 s(-1) mM(-1)) were obtained by coupling anti-LIBS to ML in a two-step post-insertion technique. We confirmed specific binding to the GPIIb/IIIa receptor's activated conformation on activated human platelets and cell lines expressing activated GPIIb/IIIa receptor ex vivo. The immuno-ML obtained in this study constitute an important step towards developing a potentially human-compatible MRI contrast agent for the timely detection of plaque rupture by targeting activated platelets.

Killing tumour cells by alkylphosphocholines: evidence for involvement of CD95.

Many lipids act as cellular messengers and lead to a variety of different cellular responses. Out of the group of these compounds the ceramides are able to induce apoptosis, and some synthetic lipids can mimic this effect. Apoptosis is an important mechanism whereby chemotherapeutics exhibit their anti-oncogenic activity. Although, some lipid analogues were used in clinical trials, they exert severe side effects and their mechanism of action is widely unknown. We present here a new class of synthetic alkylphosphocholines (APC) that induce programmed cell death in leukaemia cells. The signs of apoptosis arise after 1 h of incubation with these compounds as shown by phosphatidylserine externalisation followed by caspase activation and DNA fragmentation. We demonstrate that the molecular target of these lipids is upstream of caspases and Bcl-2. Experiments with FADD dominant negative cells reveal that induction of apoptosis occurs on the level of CD95 and that these compounds can now be optimised for their capacity to activate the apoptosis-inducing receptor CD95.

High throughput screening method for identification of new lipofection reagents.

Lipofection, the transfer of genetic material into cells by means of cationic lipids, is of growing interest for in vitro and in vivo approaches. In order to identify ideal lipofection reagents in a HTS, we have developed an automated lipofection method for the transfer of reporter genes into cells and for determination of the lipofection results. The method has specifically been designed and optimized for 96-well microtiter plates and can successfully be carried out by a pipetting robot with accessory equipment. It consists of two separate parts: (1) pretransfection (preparation of liposomes, formation of lipoplexes, and lipoplex transfer to the cells) and (2) posttransfection (determination of the reporter enzyme activity and the protein content of the transfected cells). Individual steps of the lipofection method were specifically optimized - for example, lipoplex formation and incubation time as well as cell lysis, cell cultivating, and the reporter gene assay. The HTS method facilitates characterization of the transfection properties (efficiency and cytotoxicity) of large numbers of (cationic) lipids in various adherent cell types.

The antiproliferative alkylphospholipid S-1-O-phosphocholine-2-N-acetyl-octadecane induces apoptosis in leukemia cell lines.

Lipids are involved in a multitude of important cellular functions. They act as signaling molecules and can even provoke apoptosis. In this context we investigated the efficacy of synthetic alkylphosphocholines (APCs) as potential anti-cancer membrane-affecting drugs. Leading to novel therapeutic strategies for cancer treatment, the new agents interact with the cell membrane and do not affect the DNA. The data presented here show a cell death-inducing capacity for 1-O-phosphocholine-2[S]-O-acetyl-octadecane and 1-O-phosphocholin-2[S]-N-acetyl-octadecane in Jurkat T cells as well as in BJAB cells. The activation of caspases is generally required for the induction of apoptosis as shown by experiments with specific caspase inhibitors. The results point on the one hand to the formation of a functional DISC after APC-treatment as indicated by the clustering of receptor molecules and on the other hand to the dependency on the instrinsic apoptotic machinery and the downstream of mitochondria-activated apoptosome.

Change in pharmacokinetic and pharmacodynamic behavior of gemcitabine in human tumor xenografts upon entrapment in vesicular phospholipid gels.

PURPOSE: The in vivo pharmacokinetics (PK), biodistribution and antitumor activity of a new liposomal formulation of gemcitabine (GemLip) were compared to the conventional (clinical) formulation of gemcitabine (GemConv). METHODS: Gemcitabine was entrapped in a vesicular phospholipid gel (VPG) consisting of densely packed liposomes. Redispersed VPG containing GemLip consisted of 33% liposomally entrapped and 67% free gemcitabine. The in vivo efficacies of GemLip and GemConv were compared using the subcutaneously growing human soft tissue sarcoma SXF 1301 and the orthotopically growing human bladder cancer BXF 1299T. PK and biodistribution were evaluated using radiolabeled drug and lipid in SXF 1301 tumor-bearing nude mice. RESULTS: GemLip was highly active in SXF 1301 at a gemcitabine dose of 6-9 mg/kg (days 1, 8 and 15; dose near the MTD). In the 6-mg/kg groups, complete tumor remissions were observed in seven of eight mice. Equimolar doses of GemConv resulted in only moderate tumor growth inhibition. Even at equitoxic doses (360 mg/kg given on days 1, 8 and 15, or 120 mg/kg on days 1, 5 and 8) GemConv was less active than GemLip. Furthermore, GemLip was active in the orthotopically growing BXF 1299T bladder cancer model at 6 mg/kg and prevented distant organ metastasis. In the PK study, GemLip achieved a 35-fold higher plasma AUC (1680 mg x h/ml) than GemConv (47.6 mg x h/ml). The serum half-lives were 0.15 h for free gemcitabine and 13.3 h for liposomal gemcitabine (6 mg/kg each i.v.). Moreover, gemcitabine levels in tumors were fourfold higher following injection of GemLip than following injection of GemConv. CONCLUSIONS: GemLip is a highly effective gemcitabine delivery system which results in superior gemcitabine pharmacodynamics and PK than GemConv. The enhanced in vivo efficacy might be explained by sustained release and passive tumor targeting.

Synthesis of enantiomerically pure 1-O-phosphocholine-2-O-acyl-octadecane and 1-O-phosphocholine-2-N-acyl-octadecane.

This is the first report on the chemical synthesis of enantiomerically pure R- or S-1-O-phosphocholine-2-O-acyl-octadecanes and R- or S-1-O-phosphocholine-2-N-acyl-octadecanes. From a structural point of view these phospholipids are intermediates between phosphatidylcholine and sphingomyelin. The synthesis of these model compounds is based on R- or S-1.2-O-isopropylidene-glyceraldeyde for chain elongation in a Wittig reaction with pentadecane-triphenylphosphine bromide. The resulting 1.2-O-isopropylidene-octadec-3-en is converted to R- or S-1.2-octadecanediol by catalytic hydrogenation of the double bond and by acidic removal of the isopropylidene protecting group. Tritylation of R- or S-1.2-octadecanediol results in the general intermediates R- or S-1-O-trityl-2-hydroxy-octadecane. These are the key intermediates for the synthesis of the phosphatidylcholine- or sphingomyelin-like end products. R- or S-1-O-phosphocholine-2-O-acyl-octadecane is obtained from the tritylated intermediates via benzylation in position 2, acidic detritylation and conversion of the R- or S-1-hydroxy-2-benzyl-octadecanes to the respective phosphocholines via the phosphoethanolamines. Catalytic hydrogenolysis of the benzyl group results in R- or S-1-O-phosphocholine-2-hydroxy-octadecane, which is converted to the phosphatidylcholine-like end products by acylation. R- or S-1-O-phosphocholine-2-N-acyl-octadecane is obtained from the tritylated intermediate by conversion of the R- or S-2-hydroxy group into the N-phthalimido group, which is achieved by inversion of the configuration using the Mitsunobu reaction with phthalimid. After acidic detritylation, the product is converted to the respective S- or R-1-O-phosphocholine derivative in a similar sequence of reactions. The phthalimido group is converted to the 2-amino group, and acylation results in the sphingomyelin-like end products.

A simple approach to DOTAP and its analogs bearing different fatty acids.

A simple synthesis of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP) and its analogs differing in the fatty acids is presented. The synthesis is designed as quasi-one-pot reaction and the precipitating products are purified by simple recrystallization.

Effect of nucleoside analogues and oligonucleotides on hydrolysis of liposomal phospholipids.

The hydrolysis of the bilayer forming phospholipids resulting first of all in lysophospholipids and fatty acids is one limiting factor determining the shelf-life of liposomes. In several studies the influence of pH, buffer, lipid composition and other parameters on the hydrolysis of phospholipids have been demonstrated, but the influence of drugs has not yet been investigated systematically. In this study the influence of nucleoside analogues, especially 2', 2'-difluoro 2'-deoxycytidine (gemcitabine, dFdC) on the degradation of phospholipids was elucidated in more detail. It could be demonstrated that the interaction of dFdC with phospholipid bilayers promotes the hydrolysis of phospholipids in a concentration-dependent manner. Obviously two parts of the molecule, the amino group bound to the pyrimidine moiety and the 2', 2'-difluoro-2'-deoxyribose, seem to be responsible for the forced phospholipid hydrolysis. The dFdC-induced hydrolysis of phospholipids was influenced by pH, buffer, lipid composition and different anions. Optimization of the above parameters resulted in prolonged shelf-life of dFdC liposome dispersions, which is an important prerequisite for clinical practice.

5-Fluorouracil in vesicular phospholipid gels for anticancer treatment: entrapment and release properties.

Vesicular phospholipid gels (VPG), i.e. highly concentrated liposomal dispersions, are suitable for entrapping substances such as anticancer drugs with particular high encapsulation efficiencies (EE). We prepared different formulations of VPG with 30% (w/w) lipid containing 5-fluorouracil (5-FU) by high pressure homogenization and analysed their EE and drug release. Using mixtures of hydrogenated soy phosphatidylcholine and cholesterol with molar ratios ranging from 55/45 to 75/25, a decreasing amount of cholesterol correlated with an increasing EE, which is probably due to a reduced amount of smaller vesicles and number of lamellae. Using a 5-FU solution of pH 8.6 for VPG preparation, an EE of approximately 40% was found after redispersion of the gel to a liposomal dispersion and separation of free drug from liposomal drug by size exclusion chromatography. The reduced EE for preparations with lower pH values was attributed to a fast initial drug release due to the increased drug lipophilicity below the pK(a) value of 8. After redispersion of a VPG of pH 8.0, an initially faster release of about a third of the entrapped drug was found during the first 20 min, followed by stable entrapment over many hours. The rapid initial release may be due to the portion of liposomes smaller than 40 nm in diameter, determined by photon correlation spectroscopy. Cryo electron microscopic pictures show a lentil-like shape of these small liposomes. The membrane defects on the edges are probably the reason for the very high initial drug release rate. The half-life time of the release of 5-FU from intact FU-VPG at both pH 7.4 and 8.0 was found to be in the order of 4-5 h and the kinetics are typical for matrix-controlled drug diffusion. The in vitro data of 5-FU loaded VPG suggest their applicability as implants with controlled release properties or, after redispersion, as intravenously injected liposomal formulations.

[Pharmaceutical comparability of different therapeutic Echinacea preperations]. / Zur pharmazeutischen Vergleichbarkeit von therapeutisch verwendeten Echinacea-Präparaten.

Cichoric acid and Dodeca 2E, 4E, 8Z, 10E/Z-tetraenoic acid isobutylamide (alkamides 8, 9) are described as immunomodulating active components of Echinacea. We quantified both substances in 25 Echinacea-containing remedies customary in trade. Concerning both active components, we found highly concentrated remedies as well as remedies without any detectable cichoric acid or alkamides. The concentration of both active components varied extremely depending on the type of remedy (homeopathic mother tincture, pressed juice, tablets, spagyric tincture), on the Echinacea-species (Echinacea angustifolia, Echinacea pallida, Echinacea purpurea) and on the part of the plant (root, herb, whole plant). Moreover, we found large differences between comparable drugs of different manufacturers and between different charges of the same remedy. We recommend that preclinical and clinical studies with Echinacea-containing drugs should always include the quantification of the potentially active components. We are convinced, that in the long term this will help to clarify how the different active components contribute to the immunomodulating effect of Echinacea.

[Importance of long chain omega-3 fatty acids in prostate cancer]. / Stellenwert von langkettigen Omega-3-Fettsäuren bei Prostatakrebs.

The benefits of long chain polyunsaturated omega-3 fatty acids (n-3 PUFA) from fish or administered as supplements remain controversial regarding prostate cancer (PCa). Based on the currently available evidence no clear benefit of n-3 PUFA intake to generally reduce PCa incidence has been found. On the other hand n-3 PUFAs have a clear influence on the development of already existing PCa. The intake of n-3-PUFAs considerably reduces the risk of metastasis and PCa-related mortality.