The interaction of saquinavir (soft gelatin capsule) with ketoconazole, erythromycin and rifampicin: comparison of the effect in healthy volunteers and in HIV-infected patients.

OBJECTIVE: The aim of this study was to compare the effect of ketoconazole, erythromycin and rifampicin on the pharmacokinetics of saquinavir soft-gelatin formulation (Fortovase; FTV) in healthy volunteers with that in HIV-infected patients at steady state after administration of 1200 mg three times daily. METHODS: In two open-labelled, randomised, crossover studies pharmacokinetic parameters were calculated in healthy volunteers who received on one occasion multiple doses of 1200 mg FTV three times daily alone and on the other occasion in combination with multiple doses of either 400 mg ketoconazole once daily or 600 mg rifampicin once daily. In another open-labelled, multicentre study, 33 HIV-infected patients underwent a pharmacokinetic assessment after 36-51 weeks of treatment with FTV and were then given additionally multiple doses of either 200 mg ketoconazole once daily, 250 mg erythromycin four times daily or 600 mg rifampicin once daily. Pharmacokinetic parameters of saquinavir were determined again at the end of the combination treatment. RESULTS: In healthy volunteers, coadministration of ketoconazole increased saquinavir area under the curve from time 0 to 8 h (AUC0-8 h) by 190% (95% CI: 90-343) whereas coadministration with rifampicin resulted in a decrease for AUC0-8 h by 70% (95% CI: 50-82). In HIV-infected patients, coadministration of ketoconazole and erythromycin increased AUC0-8 h of saquinavir by 69% (95% CI: 14-150) and 99% (95% CI: 33-198), respectively. When saquinavir was given together with rifampicin, exposure of saquinavir in terms of AUC0-8 h was decreased by 46% (95% CI: 18-65) compared with the baseline assessment. CONCLUSION: Interactions of saquinavir with ketoconazole, erythromycin and rifampicin were observed in healthy volunteers as well as patients. The effects observed in patients, however, appear to be less pronounced. The enzyme induction caused by rifampicin might lead to subtherapeutic levels of saquinavir and this finding appears to be of clinical relevance.

Induction of apoptosis by the O-hydroxyethyl-D(Ser)(8)-cyclosporine A derivative SDZ IMM 125 in rat hepatocytes.

The immunosuppressive cyclosporine A derivative, O-hydroxyethyl-D(Ser)(8)-cyclosporine (SDZ IMM 125), was examined for its ability to induce apoptosis in rat hepatocytes cultured for 4 or 20 h. Four hours after SDZ IMM 125 treatment, chromatin condensation and fragmentation, and the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeled and Annexin V-positive cells increased dose dependently without any observable lactate dehydrogenase leakage. The activity of the cysteine protease, caspase-3, was increased, but not that of caspase-1 and -6. The specific caspase-3 inhibitor, Ac-Asp-Glu-Val-Asp-aldehyde, inhibited caspase-3 activation and attenuated SDZ IMM 125-induced apoptosis and lactate dehydrogenase leakage. After 20 h of SDZ IMM 125 incubation, the parameters of apoptosis were further increased. Decreased mitochondrial membrane potential (measured by rhodamine 123 uptake) and cytochrome c release went in parallel with ultrastructural mitochondrial changes, and might be regarded as early events that trigger the apoptotic cascade. Transmission electron microscopy showed cytoplasmic blebbing after 4 h of SDZ IMM 125 incubation. As observed by transmission electron microscopy, treatment with SDZ IMM 125 resulted in an increase in the number of necrotic cells after 20 h, but not after 4 h. Our findings suggest that in rat hepatocyte cultures, SDZ IMM 125 is a specific inducer of apoptosis after short-term incubation, and this overlaps with necrosis after longer treatment periods. It is very likely that the necrosis occurring later is the result of the early apoptotic events.

Mechanisms of cyclosporine A-induced apoptosis in rat hepatocyte primary cultures.

In rat hepatocytes and isolated liver mitochondrial fractions, Cyclosporine A (CsA) is often used as a specific inhibitor of mitochondrial Ca(2+) release and as a specific blocker of mitochondrial membrane potential and permeability transition (MPT), which are all processes involved in the inhibition of apoptosis. However, neither inhibition nor induction of apoptosis by CsA has yet been described in the rat hepatocyte primary culture during incubation for 4 and 20 h. It was the purpose of the present study to examine by means of morphological and biochemical criteria the effects of CsA on apoptosis and to characterize the underlying mechanisms. Rat hepatocytes were cultured for 4 or 20 h with CsA at concentrations of 0, 10, 25, and 50 microM. Chromatin condensation and fragmentation, DNA fragmentation (TUNEL), membrane phosphatidylserine distribution (Annexin V), caspase-1, -3, and -6 activity, mitochondrial membrane potential (Rhodamine 123), and cytochrome c release into the cytosol were investigated. Four hours after CsA treatment, chromatin condensation and fragmentation and the number of TUNEL- and Annexin V-positive cells increased dose-dependently without any observable enzyme leakage, which indicated the integrity of the outer cell membrane. After 20 h of CsA incubation apoptosis parameters were further increased and were accompanied by the increased activity of the cysteine protease, caspase-3 (CPP 32), and slightly increased caspase-6 (Mch 2), but not caspase-1 (ICE). The caspase-3 inhibitor, Ac-DEVD-CHO, inhibited caspase-3 activation and attenuated CsA-induced apoptosis and LDH leakage. The caspase-6 inhibitor, Ac-VEID-CHO, only marginally inhibited CsA-induced apoptosis. Decreased mitochondrial membrane potential and cytochrome c release went in parallel with ultrastructural mitochondrial changes and might be regarded as early events that trigger the apoptosis cascade. Transmission electron microscopy confirmed an increase in the number of necrotic cells after 20 h, but not after 4 h, compared with controls.

Active nuclear import of single-stranded oligonucleotides and their complexes with non-karyophilic macromolecules.

The objective of this investigation was to characterize intranuclear accumulation of oligonucleotides and their adducts with non-karyophilic compounds in cultured animal cells and thus to present a model system for nucleic acid-mediated nuclear import. In digitonin-permeabilized cells, nuclear uptake of 3'-FITC-labeled, single-stranded 25-mer oligodeoxyribonucleotides was independent of added cytosolic protein, largely energy-dependent, inhibitable by wheat germ agglutinin but not by N-ethylmaleimide, and a function of their base composition. When coupled to FITC-labeled streptavidin or streptavidin-bovine serum albumin conjugates, the oligonucleotides delivered the proteins to the nuclear interior with rates roughly proportional to their karyophilicity as free molecules. Transport activity was also demonstrated for single-stranded oligoribonucleotides. The transport was energy-dependent, inhibited by GMP-PNP and wheat germ agglutinin, but unaffected by N-ethylmaleimide. Nuclear import of oligo(dG)25/protein adducts needed 3 to 4 oligonucleotide signals per complex and the signal had to be at least 15 nucleotides long. Micro-injection experiments showed that the results obtained with digitonin-permeabilized cells are not artifacts of a quasi-intact cellular system. These data were confirmed by electron microscopy employing complexes of oligodeoxyribonucleotides with streptavidin-peroxidase-bovine serum albumin-1 nm gold. In permeabilized cells, the complexes docked to the cytoplasmic face of the nuclear pore complexes, were translocated through the central pore channel and accumulated in large quantities in the nuclear baskets before they were released into the nucleoplasm. These results demonstrate that nuclear uptake of oligonucleotides and their complexes is an active process mediated by nuclear pore complexes, which, at least regarding its cytoplasmic component, is different from the pathway requiring classical nuclear localization signals.

Colocalization of single ribosomes with intermediate filaments in puromycin-treated and serum-starved mouse embryo fibroblasts.

Previous experiments have revealed a relatively weak electrostatic binding capacity of in vitro reconstituted intermediate filaments (IFs) as well as of natural IFs of whole cell mount preparations for purified ribosomal particles of mammalian origin. In order to demonstrate that such associations also occur in vivo, intact cells were subjected to double immunofluorescence microscopy using antibodies directed against vimentin and ribosomal protein S17. Since in proliferating cells the majority of the ribosomal particles are assembled into polyribosomes and these are to a great extent associated with microfilaments, in vitro cultured mouse embryo skin fibroblasts (MSF cells) were treated with puromycin to allow the formation of single ribosomes. Employing confocal laser scanning microscopy, the ribosomes were detected in colocalization with vimentin IFs. Disassembly of polyribosomes was also achieved by serum starvation of cultured cells. In this case, MSF cells of a low passage attained an extended and flattened appearance with the vimentin IFs being directly associated with the cell nuclei, radiating into the peripheral areas of the cells or showing a stress fiber-like distribution. In both cases, considerable quantities of ribosomal material were seen in close neighborhood to vimentin IFs. Frequently, these ribosome-IF associations were coaligned with microtubules and they also surrounded myosin I-decorated stress fibers. Double labeling with the vital, RNA-specific fluorochrome SYTO 14 produced a fluorescence pattern largely superimposable on that of ribosomal protein S17. Treatment of the starved cells with either demecolcine or cytochalasin D had an only moderately disturbing effect on vimentin IF distribution and the ribosomes stayed in contact with the vimentin IFs. On the basis of these results, it is conceivable that IFs play a role in the storage of ribonucleoprotein particles in general and non-translating ribosomes in particular in the cytoplasm of animal cells. In addition, the often seen coalignment of IFs with microtubules and microfilaments might serve facilitated and directional transport of ribonucleoprotein particles from the nucleus to peripheral areas of the cell.

Fluorescence microscopic comparison of the binding of phosphodiester and phosphorothioate (antisense) oligodeoxyribonucleotides to subcellular structures, including intermediate filaments, the endoplasmic reticulum, and the nuclear interior.

To detect potential intracellular binding sites for antisense oligodeoxyribonucleotides (ODN), 3'-fluorescence-tagged phosphodiester (P) and phosphorothioate (S) analogs of a series of model and vimentin and actin antisense ODN were applied to digitonin-permeabilized fibroblast and epithelial PtK2 cells. Fluorescence microscopy revealed binding of the ODN to intermediate filaments (IFs) with a preference for cytokeratin IFs, cytoplasmic membranes (endoplasmic reticulum), and, above all, the nuclear interior. The affinity of the ODN for these cellular substructures was dependent on their base composition, and the S-ODN were by far superior to the corresponding P-ODN in binding activity. Fluorescence polarization measurements of the interaction of ODN with purified IF proteins in vitro confirmed the differential, high-affinity binding of S-ODN to IFs. In permeabilized cells, the ODN readily migrated into the nucleus where, at ambient temperature, preferentially the S-ODN gave rise to a multitude of large, irregular aggregates. Nuclear uptake of the ODN was considerably and differentially inhibited by wheat germ agglutinin. High-affinity S-ODN, but not P-ODN, additionally reacted with a structure presumably identical with the nuclear lamina. Simultaneously, they cause decompaction of chromatin, whereby the S-ODN aggregates appeared as compact inclusions in homogeneously dispersed chromatin. After microinjection of S-ODN into intact cells, these effects were not observed, although the nucleic acids rapidly moved into the nucleus and condensed into a large number of well-defined, spherical speckles or longitudinal rodlets. The methylphosphonate analogs of some of the ODN used exhibited only extremely low affinities for intracellular constituents. These results show that excess amounts of S-ODN saturate a host of both low-affinity and high-affinity binding sites on cellular substructures, whereas limited quantities as used for microinjection recognize only the high-affinity binding sites. The results support the notion that the nonsequence-specific, often toxic effects of antisense S-ODN result from their strong binding to cellular components and substructures involved in replicational, transcriptional, and translational processes. On the other hand, the association of the ODN with membranes and cytoskeletal and karyoskeletal elements may serve to optimize their sequence-specific interaction with their intended target sites and also increase their cellular retention potential. These cellular structures would thus fulfill a depot function.

Binding of fluorescence- and gold-labeled oligodeoxyribonucleotides to cytoplasmic intermediate filaments in epithelial and fibroblast cells.

Previously, in vitro experiments have demonstrated the capacity of intermediate filaments (IFs) to associate with polyanionic compounds, including nucleic acids. To prove that this activity is also shown by IFs in quasi-intact cells, digitonin-permeabilized epithelial PtK2 and mouse fibroblast cells were treated with FITC-labeled, single-stranded oligodeoxyribonucleotides and analyzed, after indirect decoration of their IF systems with TRITC-conjugated antibodies, by fluorescence microscopy. While cytokeratin IFs exhibited a strong affinity for and exact codistribution with oligo(dG)25, vimentin IFs were less active in binding this oligonucleotide. Other oligonucleotides, like oligo(dT)25, oligo[d(GT)12G] and oligo[d(G3T2A)4G], were bound to IFs with lower efficiency. In general, the introduction of dA residues into oligo(dG)n or oligo(dGT)n tracts reduced the IF-binding potential of the nucleic acids. This, however, increased significantly upon reduction of the ionic strength to half physiological, indicating a strong electrostatic binding component. The binding reaction was often obscured by simultaneous association of the oligonucleotides with cellular membranes mostly in the perinuclear region, an activity that was largely abolished by prior cell extraction with nonionic detergent. Strongly IF-binding oligonucleotides also disassembled microtubules, presumably via their interaction with microtubule-associated proteins, but left microfilaments intact. In PtK2 cells, oligo(dG)25-loaded IFs were frequently seen coaligned with microfilaments and to cross-bridge stress fibers with the formation of rope ladder-like configurations. Employing microinjection and confocal laser scanning microscopy, association of IFs with oligonucleotides could also be visualized in intact cells. In accord with these fluorescence microscopic data, transmission electron microscopy of permeabilized cells treated with gold-conjugated oligonucleotides revealed decoration of IFs and membrane systems with gold particles, whereby in PtK2 cells these structures showed a distinctly heavier labeling than in fibroblasts. These results demonstrate that in animal cells IFs are able to bind nucleic acids and, very likely, also nucleoprotein particles and suggest that this capacity is exploited by the cells for transient storage and, in cooperation with microtubules and microfilaments, controlled transport of such material in the cytoplasm.

Preparation of a dansylated fibrate, a new fluorescent tool to study peroxisome proliferation. Effect on hepatic-derived cell lines.

The synthesis of a dansylated fibrate (DNS-X) has been performed in order to identify the cellular affinity sites of peroxisome proliferators and to establish the subcellular localization of such molecules. DNS-X has been obtained by coupling the dansy1 chloride with the amine resulting from the bezafibrate alkaline hydrolysis. The purified DNS-X has been further characterized by spectrum analysis (UV-Vis, fluorescence, [1H]/[13C]-NMR and mass). At 250 microM and incubated for 48 h with the rat hepatic derived cells (Fao cells), DNS-X stimulates 12-fold the palmitoyl-CoA oxidase, a peroxisome proliferation marker enzyme. This increase is comparable to the one obtained with well known peroxisome proliferators such as bezafibrate or ciprofibrate. The stimulation by DNS-X is specific for the overall molecule since neither the dansyl chloride, the amine, nor the precursors of DNS-X are active. The increase of palmitoyl-CoA oxidase activity is correlated with the increase of the enzyme amount as shown by immunoblotting. In agreement with the species-specificity of the fibrate neither DNS-X, bezafibrate nor ciprofibrate significantly increase palmitoyl-CoA oxidase activity and the enzyme amount in human hepatic-derived cells, HepG2. This work shows that the dansylated fibrate is a new fluorescent tool to study the subcellular localization and identification of high affinity binding sites, then further on, to elucidate the peroxisome proliferation mechanism and the action of hypolipidaemic agents of the fibrate family.

Separation and characterization of homo and hetero-oligomers of the intermediate filament proteins desmin and vimentin.

Affinity chromatography on single-stranded (ss)DNA-cellulose in conjunction with gel permeation chromatography in the presence of urea was employed to separate the intermediate filament (IF) protein complement of catalytically oxidized BHK-21 cell Triton cytoskeletons into disulfide-cross-linked homo- and heterodimers of desmin and vimentin and uncross-linked homodimers. The same separation was performed on a desmin-vimentin mixture under autoxidizing conditions in 6 M-urea to obtain the respective cross-linked collision complexes of both proteins. In 5 M-urea, the oxidation products were identified as dimers that were physically indistinguishable from uncross-linked homodimers, suggesting that they were in the form of partially denatured face-to face pairs. Heterodimers derived from intact IFs were identical to those derived from collision complexes. In the presence of 2-mercaptoethanol, heterodimers were unstable and transformed spontaneously into homodimers. After removal of urea, all cross-linked dimers were totally unable to polymerize into filaments; however, in the presence of 2-mercaptoethanol they showed a normal assembly competence. This inability of oxidized homo- and heterodimers to polymerize, together with the relatively low yield of cross-linked dimers obtained from cytoskeletons, is probably due to the introduction of steric strain into the dimers by disulfide bond formation. Substantial amounts of cross-linked heterodimers could also be isolated from IFs reconstituted from mixtures of desmin and vimentin in their homodimeric or tetrameric forms. Taken together, these results suggest that the cross-linked dimers isolated from cytoskeletons arise from a reaction between subfilament strands of IFs rather than from disulfide bond formation within pre-existing dimers and that the heterotypic IFs of BHK-21 cells are largely formed from homodimers and tetramers, respectively, rather than from heterodimers. The differential capacity of desmin and vimentin to interact with ssDNA has also been exploited to distinguish between homotypic and heterotypic protofilaments, the latter consisting of one homodimer of each protein species. This distinction could be made on the basis of characteristic differences in the sedimentation behavior of the respective protein-DNA complexes.