Erufosine, a novel alkylphosphocholine, in acute myeloid leukemia: single activity and combination with other antileukemic drugs.

PURPOSE: Alkylphosphocholines represent a new class of cytostatic drugs with a novel mode of action. Erufosine (ErPC3), the first compound of this class that can be administered intravenously, has recently been shown to be active against human tumor and leukemic cell lines. METHODS: In order to evaluate the antileukemic potential of ErPC3 in acute myeloid leukemia (AML) the lethal concentration 50% (LC 50) was determined using WST-1 assay. For analysis of cell death, staining for Annexin V and activated caspase 3 was performed. An interaction analysis was performed by calculation of combination index and construction of isobolograms. RESULTS: The LC 50 was 7.4 microg/ml after 24 h and 3.2 microg/ml after 72 h in HL 60 cells and 30.1 and 8.6 microg/ml, respectively, in 19 fresh samples from patients with AML. ErPC3 was found to be cytotoxic in HL60 cells with distinct activation of caspase 3. ErPC3 was not cross-resistant with cytarabine, idarubicine and etoposide as shown by the linear relation of respective LC 50s. The latter agents, however, exerted an additive cytotoxicity in combination with ErPC3 as revealed by isobologram analysis and combination index, although results are uneven for idarubicine. CONCLUSION: Based on these data ErPC3 appears as a novel antileukemic candidate drug, which needs to be explored further in the treatment of AML.

Quantification of erufosine, the first intravenously applicable alkylphosphocholine, in human plasma by isotope dilution liquid chromatography-tandem mass spectrometry using a deuterated internal standard.

A sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of erucylphosphohomocholine (erufosine, ErPC(3)) in pharmacokinetic studies. Nine-fold deuterated ErPC(3) was used as the internal standard. Following protein precipitation, reversed phase chromatography was performed. For analyte detection, electrospray ionization in the positive mode was applied. The mass transition m/z 504.4>139.1 was recorded for ErPC(3), and the transition m/z 513.7>139.1 for the internal standard, respectively. Good linearity with a correlation coefficient >0.99 was found for the range of 0.48-15 mg/L ErPC(3) in plasma (0.93-29.8 microM), the important range for clinical pharmacokinetic analysis. Interassay coefficients (n=10) of variation between 4.2% and 5.5% were found for ErPC(3) pool samples with concentrations between 4.7 mg/L and 44.0mg/L, respectively. The method has been used for analyses during a phase I clinical trial of ErPC(3).

Structural variation of cationic lipids: minimum requirement for improved oligonucleotide delivery into cells.

In vivo transfection efficiency (TE) using cationic liposome/oligonucleotide (ODN) complexes is often hampered by interactions with serum components. Novel cationic lipids with different hydroxyethyl or dihydroxypropyl ammonium backbones, esterified hydrocarbon chains and hydroxy substituents have been synthesized and applied in cationic liposome formulations with and without the helper lipid DOPE (1:1, m/m). Their properties for cellular ODN delivery were determined using fluorescently labeled ODNs (F-ODNs). Cationic lipids with hydrocarbon chains esterified to non-glycerol backbones in non-vicinal configuration were completely ineffective in nuclear ODN-delivery. Instead, an increased cytoplasmic localization of F-ODNs was observed. Cationic lipids equipped with only one hydrocarbon were completely incompetent for cellular ODN delivery. In the absence of serum, all cationic lipids tested with hydrocarbon chains in vicinal configuration esterified to a glycerol backbone (the respective N-(1,2-diacyl-dihydroxypropyl)-N,N,N-trimethyl-ammoniumchlorides or N-(1,2-diacyl-dihydroxypropyl)-N(hydroxyethyl)-N,N-dimethyl-ammoniumchlorides as well as N-(1,2-diacyl-dihydroxypropyl)-N(1,2-dihydroxypropyl)-N,N-dimethyl-ammoniumchlorides with lauroyl, myristoyl, palmitoyl, stearoyl and erucoyl chains) were able to transfect cells when combined with DOPE (20-80% nuclear fluorescence). Remarkably, only the analog esterified with two myristoyl chains was equally effective even in the absence of DOPE. By adding hydroxy groups to the N-alkyl residue, TE under serum conditions was improved yielding transfection rates of 55%, 75% and 90% for 0, 1 or 2 substituted hydroxy groups, respectively. For plasmid DNA, different requirements were identified. Again, the analog with two myristoyl chains was most effective but only in the presence of DOPE. However, the addition of hydroxy groups had no influence on the TE in the presence of serum.

Novel temperature-sensitive liposomes with prolonged circulation time.

Hyperthermia increases the efficiency of various chemotherapeutic drugs and is administered as an adjunct to chemotherapy for the treatment of cancer patients. The temperature-dependent effect can be strongly increased by the use of temperature-sensitive liposomes in combination with regional hyperthermia, which specifically releases the entrapped drug in the heated tumor tissue. The novel lipid 1.2-dipalmitoyl-sn-glycero-3-phosphoglyceroglycerol (DPPGOG), which is closely related to the naturally occurring 1.2-dipalmitoyl-sn-glycero-3-phosphoglycerol, in combination with 1.2-dipalmitoyl-sn-glycero-3-phosphocholine and 1.2-distearoyl-sn-glycero-3-phosphocholine provides long-circulating temperature-sensitive liposomes with favorable properties under mildly hyperthermic conditions (41-42 degrees C). DPPGOG facilitates temperature-triggered drug release from these liposomes (diameter, 175 nm) and leads to a substantially prolonged plasma half-life for the encapsulated drug with t(1/2) = 9.6 h in hamsters and t(1/2) = 5.0 h in rats. Quantitative fluorescence microscopy of amelanotic melanoma grown in the transparent dorsal skin fold chamber of hamsters demonstrated a favorable drug accumulation in heated tissue after i.v. application of these liposomes (42 degrees C for 1 h). The mean area under the curve for tissue drug concentration was increased by more than sixfold by application of the new liposomes compared with nonliposomal drug delivery. In summary, we present a new DPPGOG-based liposomal formulation enabling long circulation time combined with fast and efficient drug release under mild hyperthermia. This adds positively to the results with lipid-grafted polyethylenglycol used thus far in temperature sensitive liposomes and widens the possibilities for clinical applications.

Erufosine, a novel alkylphosphocholine, induces apoptosis in CLL through a caspase-dependent pathway.

The alkylphosphocholine (APC) erufosine is a synthetic phospholipid analogue with antineoplastic activity. APC are known to interact with lipid metabolism and modulate cellular signaling pathways, particularly the phosphorylation of Akt. Here, in primary CLL cells induction of apoptosis was detected with an IC50 of 22muM whereas healthy donor PBMC were less sensitive towards erufosine. Treatment with erufosine caused dose-dependent cleavage of PARP, co-incubation with caspase inhibitor z-VAD almost completely abrogated the cytotoxic effect of erufosine indicating a caspase-dependent mechanism of erufosine. Erufosine was shown to induce apoptosis in primary CLL cells and merits further investigation regarding therapeutic options in CLL.

Dual role of hexadecylphosphocholine (miltefosine) in thermosensitive liposomes: active ingredient and mediator of drug release.

Lysolipid-based thermosensitive liposomes have been successfully introduced as efficient drug delivery system with fast drug release upon heat treatment. Hexadecylphosphocholine (HePC) is structurally related to 1-palmitoyl-2-lyso-sn-glycero-3-phosphocholine (P-lyso-PC) but chemically and metabolically more stable, thereby offering strong antineoplastic, antiprotozoal and antifungal activity. We investigated the properties of HePC in low temperature sensitive (LTSL) and high temperature sensitive liposomes (HTSL) based on 1,2-dipalmitoyl-sn-glycero-3-phosphoglyceroglycerol (DPPGOG). Therefore liposomes composed of DPPC/DSPC=8:2 (molar ratio), DPPC/DSPC/DPPGOG=5:2:3, HePC/DPPC/DSPC/DPPGOG=1:4:2:3, HePC/DSPC/DPPGOG=1:6:3 and P-lyso-PC/DPPC/DSPC/DPPGOG=1:4:2:3 were prepared by the lipid film hydration and extrusion method and compared with regard to stability at 37 degrees C and the release kinetics under heating conditions (38 degrees C-45 degrees C) in the presence of fetal calf serum. Each formulation was characterized for size distribution, zeta-potential, encapsulation efficiency and phase transition temperature (T(m)). The influence of heat on the cytotoxic activity of HePC in TSL was investigated using the WST-1 assay in BFS-1 fibrosarcoma and C6 glioma cells for the low (HePC-LTSL, T(m)=42.9 degrees C) and the high (HePC-HTSL, T(m)=48.5 degrees C) temperature sensitive formulations and compared to micellar HePC or plain TSL. The cellular HePC uptake after 15 min incubation at 37 degrees C or 42 degrees C was determined by liquid chromatography tandem-mass spectrometry (LC-MS/MS). As expected, HePC increases the release rate of TSL similar to lysolipid. The cytotoxicity of HePC in TSL was heat inducible and stronger than the one induced by micellar HePC which did not respond to heat. A possible explanation for this raise in cytotoxicity of HePC in TSL is the increased cellular transfer of HePC under heating conditions demonstrated by LC-MS/MS.

In vitro characterization of phosphatidylglyceroglycerol-based thermosensitive liposomes with encapsulated 1H MR T1-shortening gadodiamide.

Thermosensitive liposomes (TSL) with encapsulated proton (1H) magnetic resonance (MR) contrast agents have been proposed for noninvasive online temperature monitoring during tumor treatment using chemotherapy combined with hyperthermia (HT). The technique exploits the fact that water exchange between the TSL interior and exterior is increased and/or the encapsulated 1H MR contrast agent is released near the gel-to-liquid crystalline phase transition temperature (Tm) of TSL and thus shortens the 1H MR relaxation time of tissue. In this work, newly developed, phosphatidylglyceroglycerol (DPPGOG)-based TSL with encapsulated 1H MR longitudinal relaxation time (T1)-shortening gadodiamide (Gd-DTPA-BMA) were characterized in vitro by measuring the temperature dependence of the T1 of these gadodiamide-containing DPPGOG-TSL samples between 30 and 50 degrees C. The measurements revealed that the T1 nonlinearly slightly decreased with increasing temperature from 30 to 37 degrees C, mainly due to increased water exchange between the gadodiamide-containing DPPGOG-TSL interior and exterior with the exception of negligible gadodiamide release. This implies that gadodiamide-containing DPPGOG-TSL were stable at temperatures < or =37 degrees C, which was also confirmed by an independent stability study. From 37 to 44 degrees C, the T1 nonlinearly markedly decreased with increasing temperature since encapsulated gadodiamide was rapidly released. Above 44 degrees C, gadodiamide was completely released and the T1 was directly proportional to temperature while heated from 44 to 50 degrees C and cooled from 50 to 30 degrees C, respectively. Additionally, gadodiamide release was theoretically quantified and this calculated concentration was consistent with the actually released amount directly obtained from the cooling course of empty DPPGOG-TSL with completely released gadodiamide.