Cell handling, membrane-binding properties, and membrane-penetration modeling approaches of pivampicillin and phthalimidomethylampicillin, two basic esters of ampicillin, in comparison with chloroquine and azithromycin.

PURPOSE: The purpose of this work was to examine and understand the cellular pharmacokinetics of two basic esters of ampicillin, pivaloyloxymethyl (PIVA) and phthalimidomethyl (PIMA), in comparison with lysosomotropic drugs (chloroquine, azithromycin). METHODS: Cell culture studies (J774 macrophages) were undertaken to study uptake and release kinetics and to assess the influence of concentration, pH, proton ionophore (monensin), and MRP and P-gp inhibitors (probenecid, gemfibrozil, cyclosporin A, GF 120918). Equilibrium dialysis with liposomes were performed to directly asses the extent of drug binding to bilayers. Conformational analysis modeling of the drug penetration in bilayers was conducted to rationalize the experimental observations. RESULTS: PIVA and PIMA showed properties in almost complete contrast with those of chloroquine and azithromycin, i.e., fast apparent accumulation and fast release at 4 degrees C as well as at 37 degrees C, saturation of uptake (apparent Kd 40 microM), no influence of monensin, MRP, or P-gp inhibitors; tight binding to liposomes (Kd approx. 40 microM); and sharp increase in calculated free energy when forced in the hydrophobic domain. CONCLUSIONS: Although they are weak organic bases, PIVA and PIMA show none of the properties of lysosomotropic agents. We hypothesize that they remain locked onto the pericellular membrane and may never penetrate cells as such in significant amounts.

Incorporation of an aldehyde function in oligonucleotides.

A nucleotide-like phosphoramidite building block that has the nucleic base replaced by the tert-butyldimethylsilyl-protected styrene glycol was synthesized. After the automatic synthesis of an oligonucleotide incorporating this synthon, the benzaldehyde function was generated by fluoride deprotection and oxidation by sodium periodate. In a similar manner, an oligonucleotide where a nucleic base was replaced by the (CH2)8CH=O chain was synthesized and conjugated with biotin derivatives.

Syntheses and hydrolysis of basic and dibasic ampicillin esters tailored for intracellular accumulation.

Readily hydrolysable basic and dibasic esters of ampicillin were synthesised by alkylation of the carboxylate function of ampicillin to obtain prodrugs that may accumulate in cells and allow for an intracellular delivery of ampicillian (Fan et al., Bioorg. Med. Chem. Lett. 1997, 7, 3107). We found that the beta-lactam ring cleavage and the hydrolysis of the ester function were competitive reactions. The prerequisite for biological activity of compounds of this type is therefore that ester hydrolysis proceeds faster than ring opening. Some synthesised compounds show promise as prodrugs since they displayed a reasonable stability and regenerate large quantities of bioactive ampicillin in broth.

Biophysical studies and intracellular destabilization of pH-sensitive liposomes.

We examined changes in membrane properties upon acidification of dioleoylphosphatidylethanolamine/cholesterylhemisuccinate liposomes and evaluated their potential to deliver entrapped tracers in cultured macrophages. Membrane permeability was determined by the release of entrapped calcein or hydroxypyrene-1,3,6-trisulfonic acid (HPTS)-p-xylene-bis-pyridinium bromide (DPX); membrane fusion, by measuring the change in size of the liposomes and the dequenching of octadecylrhodamine-B fluorescence; and change in lipid organization, by 31P nuclear magnetic resonance spectroscopy. Measurement of cell-associated fluorescence and confocal microscopy examination were made on cells incubated with liposomes loaded with HPTS or HPTS-DPX. The biophysical studies showed (i) a lipid reorganization from bilayer to hexagonal phase progressing from pH 8.0 to 5.0, (ii) a membrane permeabilization for pH <6.5, (iii) an increase in the mean diameter of liposomes for pH <6.0, and (iv) a mixing of liposome membranes for pH <5.7. The cellular studies showed (i) an uptake of the liposomes that were brought from pH 7.5-7.0 to 6.5-6.0 and (ii) a release of approximately 15% of the endocytosed marker associated with its partial release from the vesicles (diffuse localization). We conclude that the permeabilization and fusion of pH-sensitive liposomes occur as a consequence of a progressive lipid reorganization upon acidification. These changes may develop intracellularly after phagocytosis and allow for the release of the liposome content in endosomes associated with a redistribution in the cytosol.

Identification, purification and partial characterisation of an oligonucleotide receptor in membranes of HepG2 cells.

The low and unpredictable uptake and cytosolic transfer of oligonucleotides (ODN) is a major reason for their limited benefit. Improving the ODN potential for therapy and research requires a better understanding of their receptor-mediated endocytosis. We have undertaken to identify a membrane ODN receptor on HepG2 cells by ligand blotting of cell extracts with [(125)I]ODN and by photolabelling of living cells with a [(125)I]ODN-benzophenone conjugate. A major band at 66 kDa was identified by the two methods. Its labelling was saturable and competed for by unlabelled ODN of various sequences and irrespective of the presence of a phosphodiester or phosphoro-thioate backbone. This protein remained sedimentable after carbonate extraction, indicating strong membrane association. About half of the total cell amount resisted extensive surface proteolysis, suggesting a dual localisation at the plasma membrane and cytoplasmic vesicles. The protein was purified using a biotinylated ODN-benzophenone conjugate by photocrosslinking followed by streptavidin affinity purification. A sequence obtained by Edman degradation showed no homology with known proteins. Using anti-peptide antisera, labelling by western blotting revealed at 66 kDa a band with comparable properties as found by ligand blotting. Thus, a new membrane protein acting as an ODN receptor has been demonstrated.

A fluorescent radioiodinated oligonucleotidic photoaffinity probe for protein labeling: synthesis and photolabeling of thrombin.

To study the interactions between oligonucleotides and proteins, an original photoaffinity radiolabeling probe has been synthesized. Starting with a 5'-pyridyldithio-3'-amino-oligonucleotide, the photophore benzophenone was first coupled to the 3' end, through acylation by an activated ester of benzoylbenzoic acid. A fluorescein molecule was grafted by alkylation of the free 5'-SH. This compound was finally radiolabeled with 125I using IodoBeads. The selective photolabeling of thrombin in a complex protein mixture by the radioiodinated probe validates this strategy to identify oligonucleotide-binding proteins.

Is the bacteriophage lambda lysozyme an evolutionary link or a hybrid between the C and V-type lysozymes? Homology analysis and detection of the catalytic amino acid residues.

The relationship between the bacteriophage lambda lysozyme (lambda L) and the C and V-type lysozymes has been investigated by sequence alignment, secondary structure prediction and pattern recognition methods. The alignment of the amino terminal part of lambda L with that of V-type lysozymes suggests that Glu19 is a residue essential for catalysis. Its mutation to Gln leads to a completely inactive enzyme. In the alignment of the sequence of lambda L with those of the C-type lysozymes a strongly homologous fragment of about 30 amino acid residues is detected. Taking into consideration this observation and the published structural alignments between C and V-type lysozymes, a repetition of the beta-sheet motif in lambda L is proposed. The multiple alignment draws the attention to a possible catalytic role for Asp34 that would be positioned in the middle of the second strand of the beta-sheet as in the C-type lysozymes. This role is confirmed by mutagenesis. The implications of these observations in terms of the evolutionary relationship between lambda L and the other lysozymes is discussed.

Overexpression of the phage lambda lysozyme cloned in Escherichia coli: use of a degenerative mixture of synthetic ribosome binding sites and increase of the protein stability in vivo.

The R gene of the phage lambda coding for a lysozyme expressed at the end of an infection cycle in Escherichia coli has been cloned in a series of vector plasmids. Two methods for improving the efficiency of translation have been tested. First, the use of a bicistronic construction in which the ribosome binding site (RBS) of the first cistron is that of a highly expressed gene or the use of a degenerate mixture of synthetic oligonucleotides for the optimization of a RBS. The second strategy is more efficient: the analysis of a number of clones reveals that the LaL expression levels are increased by a factor between 3 and 6 times compared with the clone using the natural RBS. The expression levels are described by an approximately Gaussian histogram. The translation promoter that was found to afford the best expression (PL) is under the control of a thermolabile repressor. Under the expression conditions, the protein is partially proteolysed. The proteolysis is significantly decreased by adding salt to the growth medium. After optimization, an increase in expression by a factor of 40 is obtained compared with the initial conditions. An efficient purification protocol is described.

The 9-fluorenylmethyloxycarbonyl group as a 5'-OH protection in oligonucleotide synthesis.

Oligo-DNAs are synthesized on a solid support using the 9-fluorenylmethyloxycarbonyl group as a 5'-OH base labile protection. The synthesis of the pure protected nucleotides, a relevant phosphoramidite-type strategy of coupling, and the optimization of the deprotection steps are described. This new synthetic method is an alternative to the standard protocol that avoids acidic conditions.

Conjugates of catecholamines. 5. Synthesis and beta-adrenergic activity of N-(aminoalkyl)norepinephrine derivatives.

A novel series of N-aminoalkyl congeners and model derivatives of norepinephrine has been synthesized. Compounds that were structurally related to epinephrine were prepared from fully protected intermediates. Alternatively, isoproterenol-related compounds were synthesized via reductive amination of preformed methyl ketone derivatives with norepinephrine. The beta-adrenergic activities of these new compounds were assessed through measurement of intracellular cyclic AMP accumulation in S49 mouse lymphoma cells and displacement of iodocyanopindolol (ICYP) from membrane preparations. Congeners that contained an underivatized primary amine function exhibited virtually no activity in these assays. However, when this amine function was acylated (e.g., to an amide, carbamate, urea, sulfonamide, etc.), the products exhibited generally increased beta-adrenergic activity, which was, however, strongly dependent on the nature of the acylating group and also the length of the spacer. In particular, a benzyl carbamate derivative containing a branched, seven-carbon spacer group was 40 times more potent than isoproterenol in the in vitro S49 assay.