Hepatic distribution of a phosphorothioate oligodeoxynucleotide within rodents following intravenous administration.

The pharmacokinetics of ISIS 1082, a 21-base heterosequence phosphorothioate oligodeoxynucleotide, were characterized within rodent whole liver, and cellular and subcellular compartments. Cross-species comparisons were performed using Sprague-Dawley rat and CD-1 mouse strains. Although whole liver oligonucleotide deposition and the proportion of drug found within parenchymal and nonparenchymal cells were similar between the two rodent species as a function of both time and dose, dramatic differences in subcellular pharmacokinetics were observed. Specifically, within murine hepatocyte nuclei, drug was observed at the 10 mg/kg dose, whereas in the rat nuclear-associated levels required the administration of 25 mg/kg. Under all experimental regimens, murine hepatic nuclear-associated drug concentrations were at least 2-fold higher than those found in rat liver cells. More detailed metabolic analysis was also performed using high performance liquid chromatography/electrospray-mass spectrometry (HPLC/ES-MS) and demonstrated that although the extent of metabolism was similar for rat and mouse, the pattern of n-1 metabolites varied as a function of both species and cell type. While rat and mouse hepatocytes and rat nonparenchymal cellular metabolites were predominantly products of 3'-exonuclease degradation, mouse nonparenchymal cells contained a majority of n-1 metabolites produced by 5'-exonucleolytic activity. Based upon these data, it would appear that subcellular oligonucleotide disposition and metabolism among rodent species are more divergent than whole organ pharmacokinetics might predict.

Preparation of oligonucleotides without aldehyde abasic sites.

High-quality oligonucleotides are obtained by selective modification of sequences containing aldehyde apurinic sites with a new chromatographic tag followed by RP-HPLC separation. Hydroxylamine derivative 1 of a water soluble nonionic surfactant modifies oligonucleotides selectively at abasic sites leading to significantly increased retention.

Monitoring solution-phase combinatorial library synthesis by capillary electrophoresis.

Capillary electrophoresis has been applied to monitor model reactions in solution-phase combinatorial chemistry. In particular, the simultaneous alkylation reactions of secondary amines with a series of benzyl halides has been investigated. Reactant and product concentrations were monitored using capillary electrophoresis in a non-aqueous buffer system. The simplified sample preparation was a key feature making this an attractive method of analysis. The results demonstrate that capillary electrophoresis is a useful tool for monitoring reactions to determine initial rates, rate constants, and extinction correlation coefficients for quantitative analysis in combinatorial chemistry, and is a broadly applicable technique for the analysis of a variety of organic and bioorganic transformations.

Characterization of oligonucleotide metabolism in vivo via liquid chromatography/electrospray tandem mass spectrometry with a quadrupole ion trap mass spectrometer.

The pattern of nuclease degradation observed for an antisense phosphorothioate oligonucleotide in pig kidney was determined using liquid chromatography/electrospray mass spectrometry (LC/ESI-MS) and LC/ESI-MS/MS with a quadrupole ion trap mass spectrometer. Metabolites were separated by length using reversed-phase high-performance liquid chromatography with aqueous hexafluoropropan-2-ol-triethylamine and a methanol gradient. The individual masses of metabolites in each LC peak were determined via deconvolution and converted into potential nucleotide compositions. The nucleotide composition was used to locate metabolites within the known oligomer sequence. The identity of metabolites was confirmed using on-line LC/MS/MS to generate fragment ions suitable for sequence verification. A limited number of shorter oligonucleotide fragments were observed, suggesting that metabolism in vivo may be sequence dependent.

On-line HPLC electrospray mass spectrometry of phosphorothioate oligonucleotide metabolites.

Metabolism of 2'-deoxyphosphorothioate oligonucleotides ISIS 11061 and ISIS 11637 was examined with capillary gel electrophoresis (CGE) and on-line HPLC electrospray mass spectrometry (HPLC/ES-MS). Oligonucleotides were isolated from plasma, liver, and kidneys of rats injected with ISIS 11061 and ISIS 11637. Metabolites found in plasma were consistent with 3'-exonuclease activity. Metabolites isolated from liver and kidney were consistent with 3'- and/or 5'-exonuclease activity. HPLC/ES-MS analysis of ISIS 11061 isolated from kidney indicated extensive degradation from the 3' terminus, but metabolites consistent with 5' degradation and combinations of 3' and 5' truncations also were observed. ISIS 11061 isolated from liver showed less extensive degradation. The 5' truncated metabolites represented the predominant species in contrast to the kidney sample. Metabolites with masses consistent with combinations of 3' and 5' truncations were also observed in liver. The metabolic profiles generated by CGE analysis of these samples agreed qualitatively with mass spectrometric results. HPLC/ES-MS enabled the simultaneous determination of degradation products that are the same length but differ in composition. CGE could discriminate species that differed by one nucleotide in length. HPLC/ES-MS was shown to be a useful tool to study the complex metabolism of antisense oligonucleotides in vivo.

Negative ionization micro electrospray mass spectrometry of oligonucleotides and their complexes.

The utility of negative ionization micro electrospray (microspray) mass spectrometry is demonstrated for detection of oligonucleotides and their non-covalent complexes. A simple microspray ionization source is fabricated from an outer stainless-steel needle and an inner fused-silica capillary. Under these conditions, the liquid flow rate can be reduced 15-fold from 7.5 microL/min to 0.5 microL/min. Studies of a 14-mer DNA oligonucleotide show no change in the charge-state distribution and quantity of adducted salt ions during the microspray process compared to pneumatically assisted electrospray mass spectrometry. The microspray ion source is less sensitive to the presence of solution buffers, and an 11-fold increase in integrated ion abundance from oligonucleotide analyte is observed with a 10 mM concentration of ammonium acetate, compared to pneumatically assisted nebulization (PAN). A > 100-fold increase in the duplex:single strand ratio for a 14-mer oligodeoxynucleotide and its complementary strand is observed using the microspray ion source relative to experiments performed with PAN. Studies of duplexes between DNA and a peptide nucleic acid suggest that this effect may be related to the degree of adduction of counterions to the DNA during ionization.

Superantigen-reactive T cells that display an anergic phenotype in vitro appear functional in vivo.

Clonal deletion and/or inactivation establishes tolerance to self antigens. Endogenous and exogenous (bacterial) superantigens, like the staphylococcal enterotoxins, induce ligand-specific clonal anergy in vivo and thus are believed to mirror aspects of post-thymic tolerance mechanisms in mature peripheral T cells. Here we analyzed the level of anergy of ligand-responsive V beta 8+ T cells from staphylococcal enterotoxin B (SEB)-primed mice in vivo and in vitro. Upon in vitro restimulation with SEB, CD4+V beta 8+ and CD8+V beta 8+ T cells failed to produce IL-2. However, functional IL-2 receptors were triggered, since supplementation with IL-2 induced clonal growth in virtually all CD4+V beta 8+ and CD8+V beta 8+ T cells as determined by limiting dilution analyses. Thus in vitro unresponsiveness of lymphocytes from SEB-primed mice reflects the inability of SEB-reactive V beta 8+ T cells to produce IL-2. Surprisingly, anergy as defined in vitro was at variance with that in vivo. Following further challenge with SEB, systemic and acute lymphokine production (including IL-2 and tumor necrosis factor) occurred with almost identical peak values and kinetics to primary in vivo responses, and D-galactosamine-sensitized mice succumbed to lethal shock. Polymerase chain reaction analyses revealed that CD4+V beta 8+ expressed IL-2-specific mRNA in vivo upon restimulation with SEB. While lymphokine production and expression of the IL-2 receptor was similar to the response to in vivo primary stimulation, only CD8+V beta 8+ T cells expanded clonally upon reintroduction of SEB in vivo. Hence primed V beta 8+ T cells challenged with SEB display in vitro anergy yet in vivo responsiveness, at least in part. We conclude that the state of anergy is reversible, dependent upon the quality of activation signals provided in in vivo rather than in in vitro culture conditions.

Superantigen-induced anergy of V beta 8+ CD4+ T cells induces functional but non-proliferative T cells in vivo.

The response profile of staphylococcal enterotoxin B (SEB)-primed murine V beta 8+ CD4+ and V beta 8+ CD8+ T cells was analysed upon rechallenge in vitro. While in vitro responses to secondary stimulation with SEB were reduced to background levels, the in vivo reactivity after rechallenge with SEB was retained, in that SEB-primed mice succumbed to lethal T-cell shock, lymphokines [interleukin-1 (IL-1), IL-2, Il-4, IL-6, IL-10, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha)], and lymphokine-specific mRNA accumulation could be detected in V beta 8+ CD4+ and V beta 8+ CD8+ T cells. However, V beta 8+ CD4+ T cells failed to enter the cell cycle. While the phenotype of V beta 8+ CD8+ T cells was indistinguishable from that of their counterparts from naive mice, V beta 8+ CD4+ T cells exhibited in vivo an unusual phenotype as non-proliferative but functional T cells. We conclude that in vitro-defined anergy does not disclose the functional abilities of ligand-reactive V beta 8+ T cells in vivo, and that priming with superantigen (SAg) induces in vivo a differentiation of SEB-reactive V beta 8+ CD4+ T cells into a non-proliferative but functional phenotype.

Analysis of double-stranded oligonucleotides by electrospray mass spectrometry.

Double-stranded oligonucleotides of different lengths and chemical modification have been analyzed by ion spray mass spectrometry. The non-covalent-bonded duplexes can be detected. Therefore, ion spray mass spectrometry is a useful method for investigation of hybridizations of natural and chemically modified oligonucleotides. Since the exact mass of the double strand can be detected, this method can distinguish between specific and nonspecific interaction.

Exogenous superantigens acutely trigger distinct levels of peripheral T cell tolerance/immunosuppression: dose-response relationship.

Ligand-specific immunosuppression requires an understanding of the parameters that control peripheral T cell tolerance. T cell receptor (TcR) transgenic mice offer a clear advantage for studying post-thymic tolerance mechanisms in vivo that are operational in a monoclonal T cell population with preselected antigen specificity. Yet it is unclear whether the rules defined in monoclonal T cells of genetically manipulated mice reflect those operative in clonally diverse peripheral T cells of normal mice. To analyze acute tolerance mechanisms in unselected peripheral T cells, we challenged normal mice with the superantigen staphylococcal enterotoxin B (SEB) and analyzed ligand-reactive V beta 8+ T cells for TcR-triggered tolerance mechanisms such as anergy, TcR down-regulation, or apoptosis. Upon challenge with graded doses of SEB (0.001-10 micrograms) V beta 8+ T cells become anergic within 6-16 h. Importantly, a dosage effect of SEB in regard to the level of anergy induced was observed. Anergy induced by low concentrations of SEB (0.001-0.1 microgram) is transient and is overcome by clonal growth, while higher concentrations of SEB (0.1-10 micrograms) cause long-lasting anergy resistant to cell cycle progression. At high SEB concentrations (1-10 mg) about 50% of the anergic V beta 8+ T cells additionally down-regulate their TcR-CD3 complex, followed by a loss of CD2, CD4, CD8 accessory molecules. In parallel, T cell phenotype-negative but genotypically V beta 8+ T cells are generated. The T cell phenotype-negative cells reacquire their V beta 8+ T cell phenotype upon culture in vitro. In vivo, a subset of V beta 8+ cells, defined by an intermediate stage of TcR down-regulation, i.e. V beta 8lowCD3+ cells, but not T cell phenotype-negative cells are selectively programmed for apoptosis, which occurs within 1 h. These data suggest that SEB triggers distinct tolerance pathways which operate in a hierarchical fashion in clonally diverse ligand-reactive T cells. Specifically, the results illustrate the power of exogenous superantigens to exploit these distinct tolerance pathways, thereby achieving distinct levels of immunosuppression.

Superantigen mediated shock: a cytokine release syndrome.

Treatment of animals with superantigens results in profound immunological changes. A major fraction of all peripheral T cells becomes activated in vivo. Subsequently, successive waves of cytokines are produced with TNF playing a central pathophysiologic role. In addition, if the liver is damaged by an as yet poor defined mechanism the consequences of the cytokine syndrome are life threatening. However, TNF alone is not sufficient to cause death, instead synergizing interactions with cytokines like IL-1, IL-6, and IFN-gamma are probably involved. On the other hand, certain experimental conditions prevent these waves of cytokines and consequently lethal shock. Furthermore, a significant fraction of SA reactive T cells are deleted by programmed cell death 10 to 24 hours after treatment. Thereafter the surviving cells proliferate vigorously until day 2 or 3, followed by a second wave of apoptosis resulting in reduced SA reactive T cell numbers as compared to pretreatment levels. Of course, many aspects of the complicated events are only marginally understood and deserve further investigation.

Optimization of capillary electrophoresis of mixtures of basic peptides and comparison with HPLC.

Fourteen synthetic, strongly basic hexadeca- to octadecapeptides, which differ only in one amino acid residue, were synthesized and characterized. In order to investigate the application of capillary electrophoresis for the separation of mixtures of closely related basic peptides, seven different mixtures, which represent possible byproducts during solid-phase synthesis, were analyzed by HPLC and different techniques of capillary electrophoresis. Peaks within the mixture were identified by single peak injection or on-line CE/MS. Utilization of micellar electrokinetic capillary electrophoresis offered the separation of complex mixtures. Comparing the electrophoretic mobilities with calculated correlations, we found that the separation of peptides of this length is influenced not only by the different size and charge but also by hydrophobicity and the orientation of the charge due to different conformations.

Separation of cardiac glycosides by micellar electrokinetic capillary electrophoresis.

The separation of mixtures of primary and secondary cardiac glycosides by micellar electrokinetic capillary electrophoresis modified by cyclodextrins, urea and sodium cholate proved to be suitable for the determination of these hydrophobic compounds. It was possible to distinguish the two anomeric cardenolides glucodigifucoside and glucodigiglucomethyloside with all three buffer systems. Electropherograms of crude plant cell extracts from Digitalis lanata were obtained with this method.

Quantitative analysis of lymphokine mRNA expression by a nonradioactive method using PCR and anion exchange chromatography.

Amplification of DNA by the polymerase chain reaction (PCR) has become an efficient tool in the study of gene expression. We describe the use of HPLC anion exchange chromatography to quantitate PCR products amplified from cDNA. The technique circumvents the use of both radioactivity and gel electrophoresis. We show that the method permits accurate quantitation of the gene product of interest and provides a clear separation of specific and non-specific products. The technique was applied to quantitate TNF-beta mRNA levels in unstimulated and stimulated mouse T cells.