Cell-cycle arrest and p53-independent induction of apoptosis in vitro by the new anticancer drugs 5-FdUrd-P-FdCydOct and dCydPam-P-FdUrd in DU-145 human prostate cancer cells

 Purpose: Current therapies have limited impact on the progression of metastatic hormone-refractory prostate cancer. Therefore, we investigated the utility of new heterodinucleoside phosphate dimers of 5-fluorodeoxyuridine (5-FdUrd) in p53-mutated and androgen-independent DU-145 human prostate tumour cells. Methods: The effects of the dimers were assessed in vitro by a cell proliferation assay for cytotoxicity, flow cytometry for cell cycle distribution, confocal laser scanning microscopy for the detection of apoptotic bodies, poly(ADP-ribose) polymerase cleavage for caspase 3 activity and by a thymidylate synthetase assay. Results: The new dimers N ⁴-palmitoyl-2′-deoxycytidylyl-(3′→5′)-5-fluoro-2′-deoxyuridine (dCydPam-P-FdUrd) and 2′-deoxy-5-fluorouridylyl-(3′→5′)-2′-deoxy-5-fluoro-N ⁴-octadecylcytidine (5-FdUrd-P-FdCydOct) caused marked cytotoxicity with IC₅₀ values of 3–4 μM. 5-FdUrd-P-FdCydOct at 200 μM was capable of eradicating 100% of tumour cells whereas 10% of the cells were resistant to 5-FdUrd. Cytotoxicity was caused by a dramatic S-phase arrest, resulting in an increase of this cell population from 34% to 85% with 5-FdUrd-P-FdCydOct and to 81% with dCydPam-P-FdUrd. S-phase arrest was followed by apoptosis, as shown by 85% of the cells staining positive for Apo 2.7 antibody, a six- to eight-fold increased caspase 3 activity and DNA fragmentation. Thymidylate synthase activity was inhibited by 50% at 0.6–0.7 μM dimer concentration. The dimers were hydrolysed in vitro by phosphodiesterase I and human serum to the corresponding nucleosides and nucleoside monophosphates. Conclusions: The new dimers dCydPam-P-FdUrd and 5-FdUrd-P-FdCydOct are effective prodrugs of 5-FdUrd and have potential value for the treatment of p53-mutated and hormone-independent human prostate carcinomas. Received: 20 August 1999 / Accepted: 4 December 1999     

Liposomes and influenza viruses as an in vitro model for membrane interactions I. Kinetics of membrane fusion and lipid transfer

To study membrane interactions, we use a previously described model system with intact PR8 influenza viruses and small liposomes containing the virus receptor G_D1a. The assay is based on the fluorescent membrane marker octadecylrhodamine B chloride, R18, incorporated in liposomes. With this assay two processes can be studied in parallel: hemagglutinin-dependent virus/liposome fusion and spontaneous marker transfer. We present a detailed kinetic analysis of fusion and transfer which takes into account the contribution of dequenching in donor and acceptor vesicles, respectively. Both fusion and transfer follow second-order kinetics; they differ, however, in the interacting species. Fusion is collision-mediated, i.e. it depends on the total particle concentration. For R18 transfer, the rate is independent of the particle concentration, but determined by the R18 surface density, i.e. the R18/lipid ratio, which is responsible for the initial quenching of the labelled species.     

Assessment of thrombogenic potential of liposomes

The effects of liposomes with positive, neutral and negative surface charges on platelets and the plasmatic coagulation system were investigated in several in vitro and in vivo models. Negatively charged liposomes stimulated the plasmatic contact activating system as demonstrated by significant acceleration of whole blood clotting time measured in containers with nonwettable (siliconized) surface. The same liposomes induced reversible aggregates of human platelets in vitro and circulating reversible platelet aggregates after intravenous infusion in guinea pigs. Liposomes with positive and neutral surface charges had no effect on plasmatic coagulation and platelets. The biological mechanisms and the toxicological relevance of these findings are discussed.     

MDCK Cell Cultures as an Epithelial In Vitro Model: Cytoskeleton and Tight Junctions as Indicators for the Definition of Age-Related Stages by Confocal Microscopy

Purpose. Madin Darby Canine Kidney (MDCK) cells were grown in culture, and age-related morphological changes in the cytoskeleton and tight junction (TJ) network were used to define stages in view of establishing an optimal in vitro model for the epithelial barrier. Methods. Growth curves and transepithelial electrical resistance (TEER) were determined, and the cytoskeleton (actin, -tubulin, vimentin) and TJ (Zonula occludens proteins ZO1, ZO2) were investigated with immunofluorescent methods by confocal laser scanning microscopy (CLSM) and digital image restoration. Results. TEER measurements indicated that TJ were functional after one day. Values then remained constant. Four morphological stages could be distinguished. Stage I (0–1 day): Sub confluent cultures with flat cells; TJ established after cell-to-cell contacts are made. Stage II (2–6 days): Confluent monolayers with a complete TJ network, which remains intact throughout the later stages. Stage III (7–14 days): Rearrangement in the cytoskeleton; constant cell number; volume and surface area of cells reduced (cobble-stone appearance). Stage IV ( 15 days): Dome formation, i.e. thickening and spontaneous uplifting of the cell monolayer. Conclusions. Based on the structural characteristics of stage III cell cultures, which are closest to the in vivo situation, we expect them to represent an optimal in vitromodel to study drug transport and/or interactions with drugs and excipients.     

Permeation Studies In Vitro and In Vivo of Potential Radiopharmaceuticals with Affinity to Neuro Receptors

Purpose. To check the influence of structuralcharacteristics on theirpermeation through the blood—brain barrier (BBB), a set of radioactive[^99mTc]chelates bearing amine groups was synthesizedand tested in vitro as well as in vivo. Methods. Compounds with different log P and pK_a values wereobtained by complex forming reactions of [^99mTc]pertechnetate withvarying substituents. Transport was studied in rats and mice, as wellas in an ECV304 cell culture model. Results. In vitro higher permeation was found for compounds withelectron attracting substituents in -position to the amine group (pK_avalues 7.4 to 8.3) than for those with more basic amine groups (pK_avalues > 8.9) even for similar log D _{pH 7.4}. In vivo brain uptake between0.8 and 4.8;pc of the injected dose (ID) per organ was found for theformer, whereas <0.4% ID were present for the latter. Conclusions. Three structurally diverse classes of [^99mTc]chelatesshowed distinct patterns with regard to brain uptake in vivo and BBBpermeability in vitro which could not be predicted by their lipophilicityalone. The close correlation between the data from rats and mice andthose obtained with cell cultures render the ECV304 cells an attractivemodel for the screening of new compounds.     

The pH-Dependence in the Partitioning Behaviour of (RS)-[3H]Propranolol Between MDCK Cell Lipid Vesicles and Buffer

Purpose. The pH-dependent partitioning of (RS)-[³H]propranolol between unilamellar vesicles of MDCK cell lipids and buffer was determined. Methods. Partitioning studies were performed by means of equilibrium dialysis at 37°C between pH 7 and 11 at a molar propranolol/lipid ratio in the membrane of 10^–6. Results. The partition-pH diagram was bell-shaped. The highest apparent partition coefficient was 1797 at pH 9.7, the lowest was 805 at pH 6.9. Curve fitting with a combination of Henderson-Hasselbalch equations revealed an inflection point at the apparent pK_a of proprano-lol, i.e. 9.7, and two additional pK_a values at pH 7.7 and 10.0. The first one corresponds to the pK_a of free fatty acids (FFA) within lipid bilayers and the other one to the pK_a of phosphatidylethanolamine (PhE). The true partition coefficients (P) of the neutral as well as the ionised solute were fitted for each ionisation status of the membrane. The highest P, i.e. 2123, was calculated for neutral propranolol in the membrane with deprotonated FFA and protonated PhE. Conclusions. The partitioning behaviour of (RS)-[³H]propranolol in a complex membrane/buffer system can be described when considering ionisation changes of drug and lipids.     

Pharmacokinetic studies of mitoxantrone and one of its metabolites in serum and urine in patients with advanced breast cancer

Objective: Mitoxantrone (MTO) was administered to patients with advanced breast cancer either as free MTO (f-MTO) or liposomal MTO (l-MTO). The intra- and interindividual variations in serum pharmacokinetics of MTO were analysed. In addition, the excretion of MTO and its metabolite mitoxantrone dicarboxylic acid (MTOD) in urine was determined. Methods: The concentration of MTO was measured by high-performance liquid chromatography in serum over a period of 24 h and the amount of MTO and the metabolite MTOD excreted in urine over 18 h was determined. Pharmacokinetic parameters of f-MTO and l-MTO were calculated. Results: l-MTO had a significantly longer half-life of distribution in the deep (third) compartment and thus a larger area under the curve (AUC) than f-MTO. No difference was found with respect to distribution in the peripheral (second) compartment. The kinetics of MTO in serum did not significantly differ between patients. In four patients repeated pharmacokinetic analyses gave superimposable results. Thus, there was no enzyme induction during therapy. By contrast, two patients with oedema had a much longer mean residence time (MRT) and AUC for MTO in serum. Despite the altered pharmacokinetics of f-MTD and l-MTO, no toxic adverse effects occurred in these two patients. Conclusions: f-MTO and l-MTO exhibited different distribution patterns in the deep compartment with a significantly increased half-life for l-MTO. There is no need to monitor MTO for treatment of breast cancer patients with f-MTO. In patients with oedema, the MRT of MTO is prolonged. The clinical relevance of this observation is as yet unclear. Received: 14 April 1997 / Accepted in revised form: 11 December 1997     

Comparing the Lipid Membrane Affinity and Permeation of Drug-like Acids: The Intriguing Effects of Cholesterol and Charged Lipids

Purpose Lipid bilayers regulate the passage of solutes into and between cellular compartments. A general prerequisite for this passage is the partitioning of the solute into the bilayer. We investigated the relationship between bilayer partitioning and permeation of three drug-like acids in liposomal systems consisting of phosphatidylcholine alone or mixed with cholesterol or charged lipids. Materials and Methods Bilayer partitioning was determined by equilibrium dialysis. Bilayer permeation was studied with a luminescence assay which is based on the energy transfer of the permeant to intraliposomal terbium(III). Results The influence of the lipid composition on the pH-dependent membrane affinity was in accordance with the membrane rigidity and possible electrostatic interactions between the acids and the lipids. However, there was no direct relationship between membrane affinity and permeation. This seeming discrepancy was closer analyzed with numerical simulations of the permeation process based on the single rate constants for partitioning and translocation. The simulations were in line with our experimental findings. Conclusions Depending on the single rate constants and on the geometry of the system, lipid bilayer permeation may positively, negatively or not correlate with the bilayer affinity of the permeant. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Isolated Rafts from Adriamycin-Resistant P388 Cells Contain Functional ATPases and Provide an Easy Test System for P-glycoprotein–Related Activities

No HeadingPurpose. P-glycoprotein (P-gp), a membrane ATPase expelling many structurally unrelated compounds out of cells, is one of the major contributors to multidrug resistance. It is enriched in cold TritonX-100 insoluble membrane domains (i.e., rafts). The purpose of this work was to characterize the ATPase activities of raft preparations from P388 cells overexpressing P-gp (P388/ADR) or devoid of P-gp (P388) and to establish a P-gp–enriched screening system for P-gp–interfering compounds.Methods. Rafts were extracted with cold TritonX-100. The ATPase activity was characterized in 96-well plates using a fluorescence assay.Results. The ATPase activity per mg protein was about five times higher in P388/ADR rafts than in crude membranes. The anti–P-gp antibody C219 inhibited 20% of the activity in P388/ADR rafts but only about 10% of the activity in P388/ADR crude membranes and had no effect on the activity of P388 rafts. The known P-gp–activating compounds verapamil, progesterone, and valinomycin revealed the typical bell-shaped activity/concentration profiles in P388/ADR rafts, indicative for activation at low compound concentrations and inhibition at concentrations >10 to 100 M. The inhibitory effect was also observed in P388 rafts.Conclusions. Extracted rafts are rich in functional ATPases. Rafts from P-gp–overexpressing cells display P-gp–typical ATPase activity and provide an easy, P-gp–enriched screening system.