Jamal Temsamani

Pharmacokinetics of Antisense Oligonucleotides

SummaryAntisense oligonucleotides are promising therapeutic agents for the treatment of life-threatening diseases.Intravenous injection of phosphodiester oligonucleotide analogue (P-oligonucleotide) in monkeys shows that the oligonucleotide is degraded rapidly in the plasma with a half-life of about 5 minutes. Administration of a single dose of the phosphorothioate (S-oligonucleotide) in animals by the intravenous route reveals biphasic plasma elimination. An initial short half-life (0.53 to 0.83 hours) represents distribution out of the plasma compartment and a second long half-life (35 to 50 hours) represents elimination from the body. This elimination half-life was similar when the oligonucleotide was administered subcutaneously. In contrast, methylphosphonate oligonucleotides have an elimination half-life of 17 minutes in mice.S-Oligonucleotide was distributed into most of organs of rats and mice. Liver and kidney were the 2 organs with highest uptake of the oligonucleotide. The S-oligonucleotide was primarily excreted in urine. Up to 30% was excreted in the first 24 hours.Repeated daily intravenous injections of a 25-mer S-oligonucleotide into rats showed that the concentrations in the plasma are at steady-state during the 8 days’ administration.The data represented here support the potential utility of phosphorothioate and methylphosphonate oligonucleotides as therapeutic agents in vivo.

Effect of different chemically modified oligodeoxynucleotides on immune stimulation

Based on previous studies that certain oligonucleotides can stimulate cell proliferation and immunoglobulin production, this study was carried out to establish the relationship between the stimulatory effect and the chemical modification of the oligonucleotide. First, the effects of oligonucleotide and analogs on immune stimulation were studied in vitro using murine splenic lymphocytes. Our results show that cell proliferation and immunoglobulin production (IgG and IgM) depend on the sequence and the chemical modification of the oligonucleotide. Phosphorothioate oligodeoxynucleotides displayed a greater stimulatory effect than partially modified phosphorothioate oligonucleotides. Second, we studied the effects of these chemically modified oligonucleotides after injection in mice. Massive splenomegaly and stimulation of cell proliferation were observed with some phosphorothioate oligonucleotides. These effects were minimized markedly by chimeric and hybrid oligonucleotides. We also demonstrate that in vitro the effects of oligonucleotides on murine lymphocytes were unaffected by T cell depletion, suggesting that oligonucleotides exert their effects mainly on the B cells.

Pharmacokinetics of an anti-human immunodeficiency virus antisense oligodeoxynucleotide phosphorothioate (GEM 91) in HIV-infected subjects

Human pharmacokinetics of an antisense oligodeoxynucleotide phosphorothioate (GEM 91) developed as an anti—human immunodeficiency virus (HIV) agent was carried out in this study. 35S‐Labeled GEM 91 was administered to six HIV‐infected individuals by means of 2‐hour intravenous infusions at a dose of 0.1 mg/kg. Plasma disappearance curves for GEM 91—derived radioactivity could be described by the sum of two exponentials, with half‐life values of 0.18 ± 0.04 and 26.71 ± 1.67 hours. The radioactivity in plasma was further evaluated by polyacrylamide gel electrophoresis, showing the presence of both intact GEM 91 and lower molecular weight metabolites. Urinary excretion represented the major pathway of elimination, with 49.15% ± 6.80% of the administered dose excreted within 24 hours and 70.37% ± 6.72% over 96 hours after dosing. The radioactivity in urine was associated with lower molecular weight metabolites. No drug‐related toxicity was observed.

Modulation of oligonucleotide-induced immune stimulation by cyclodextrin analogs

Some phosphorothioate oligonucleotides have been shown previously to stimulate cell proliferation and immunoglobulin production. In the current study, we examined the effects of cyclodextrin analogs as immunomodulatory agents for oligonucleotide-induced immune stimulation, both in vitro and in vivo. Incubation of splenocytes with a 27-mer phosphorothioate oligonucleotide that induces immune stimulation increased cell proliferation as measured by [3H]thymidine incorporation, whereas treatment of splenocytes with the phosphorothioate oligonucleotide complexed to cyclodextrin analogs markedly reduced oligonucleotide-induced cell proliferation. Similarly, administration of the 27-mer phosphorothioate oligonucleotide into mice resulted in splenomegaly and an increase in IgM production 48 hr post-administration. Administration of the oligonucleotide along with cyclodextrin analogs resulted in a significant suppression of splenomegaly and IgM response. Such suppression was dependent on the concentration of cyclodextrin analogs and was observed with various other immune stimulatory phosphorothioate oligonucleotide sequences. Administration of cyclodextrin analogs alone had no effect on splenomegaly or immune stimulation.

Effect of G-rich sequences on the synthesis, purification, hybridization, cell uptake, and hemolytic activity of oligonucleotides

Abstract We designed G-rich oligonucleotides in which the position and length of the guanosine residues were modified and studied the effects of these contiguous guanosine residues on the synthesis, purification, hybridization, cell uptake, and hemolytic effect of oligonucleotides. Our results revealed that the hyperstructure formation of these oligonucleotides depended on the length and position of the guanosine residues and the flanking sequences. The phosphorothioate oligonucleotides formed much less hyperstructures than their phosphodiester counterpart. These hyperstructures were more stable towards nucleases, were taken up by cells efficiently, and showed pronounced polyanionic related side-effects. Several phosphodiester and phosphorothioate G-rich oligonucleotides were synthesized and evaluated in respect of their ability to form hyperstructures and their hybridization characteristics. Additionally, their cell uptake and hemolytic activity were studied.

Capped Oligodeoxynucleotide Phosphorothioates.: Pharmacokinetics and Stability in Mice

Antisense oligonucleotide phosphorothioates offer a chemotherapeutic approach to acquired immunodeficiency syndrome (AIDS) by selectively blocking the expression of genes from HIV, the causative agent of AIDS.’ The oligonucleotide phosphorothioates are effective in inhibiting HIV replication in tissue culture. A pharmacokinetic study of oligonucleotidk phosphorothioatesz showed that oligonucleotide was distributed throughout most tissues and was quite stable in most organs except the liver and kidney. In the liver and kidney, extensive degradation of oligonucleotide phosphorothioate was observed. In the present study, various “end capped” oligonucleotide phosphorothioates were synthesized (TABLE 1) and studied for their comparative stability in monkey plasma (FIG. 1) and their pharmacokinetics and in vivo stability in mice. Synthesis of the oligonucleotide phosphorothioate was carried out with H-phosphonate chemistry using an automated synthesizer. 3’-, 3’,5’-, and 5‘-modifications on oligonucleotide phosphorothioates were carried out using N-Fmoc-o-DMTr-3amino-1,2-propanediol reagent either as H-phosphonate for 5’-end modification or attached to CPG for 3’-end modification. After assembly of the required sequence, CPG-bound oligonucleoside H-phosphonate was oxidized with elemental sulfur to generate phosphorothioate internucleotide linkages. Oligonucleotide phosphorothioates were deprotected and purified as reported earlier.3 %.-labeled end-capped oligonucleotide phosphorothioates were synthesized by oxidizing the CPG-bound oiigonucleoside H-phosphonate with 3sS~2. Study of the stability of “end-capped’’ oligonucleotide phosphorothioates in monkey plasma (FIG. 1) showed that 3’-end capping of oligonucleotide phosphorothioate protected the oligonucleotides from degradation, whereas 5’-end capping failed to do so. This indicates that degradation of oligonucleotide phosphorothioate is attributable mainly to 3’-exonucleases. End capped oligonucleotide phosphorothioates were studied for their pharmacokinetics and stability in mice. A single dose of 30 mg/kg was administered intravenously. The rate of excretion was about 30% at 24 hours postdosing, independent of end-capped modification. Analysis of the stability of oligonucleotide phosphorothioate excreted in the urine by polyacrylamide gel electrophoresis showed that uncapped and 5 ‘-capped oligonucleotide phosphorothioates were degraded extensively, whereas 3’-capped and 3’,5’-capped oligonucleotide phosphorothioate remained almost intact as 20 mers after 24 hours postdosing.

Enzymatic labeling of nucleic acids

Enzymatic labeling of nucleic acids is a fundamental tool in molecular biology with virtually every aspect of nucleic acid hybridization technique involving the use of labeled probes. Different methods for enzymatic labeling of DNA, RNA and oligonucleotide probes are available today. In this review, we will describe both radioactive and nonradioactive labeling methods, yet the choice of system for labeling the probe depends on the application under study.

Peptide-vector strategy bypasses P-glycoprotein efflux, and enhances brain transport and solubility of paclitaxel.

We present the results obtained with paclitaxel coupled to a peptide-vector SynB3 (PAX-OSUC-SynB3), showing that this peptide-vector enhances the solubility of paclitaxel and its brain uptake in mice using the in situ brain perfusion model. We also show by the in situ brain perfusion in P-glycoprotein (P-gp)-deficient and wild-type mice that vectorized paclitaxel bypasses the P-gp present at the luminal side of the blood–brain barrier. The effect of the vectorized paclitaxel on various cancer cells was not significantly different from that of free paclitaxel. These results indicate that vectorization of paclitaxel may have significant potential for the treatment of brain tumors.

Comparative pharmacokinetics of antisense oligonucleotides.

Antisense oligonucleotides have attracted special interest as a novel class of therapeutic agents for the treatment of viral infection, cancers, and genetic disorders because of their ablhty to inhibit expression of a disease-associated gene in a sequence-specific manner. Gene expression is inhibited by hybrid ization of the oligonucleotide to sequences in the gene or the messenger RNA (mRNA) target by Watson-Crick base pairing. The first example of specific mhibition of gene expression by an ohgonucleotide was reported by Zamecnik and Stephenson (1), who demonstrated that a short oligonucleotide inhibited Rous sarcoma virus replication in cell culture. Since then, the field has progressed enormously. Numerous studies have demonstrated the ability of antisense oligonucleotides to modulate gene expression (2-4). Accompanying chapters in this volume describe the use of antisense oligonucleotides for vanous disease targets.

Synthesis of di-, tri-, and tetrameric building blocks with novel carbamate internucleoside linkages and their incorporation into oligonucleotides

Abstract Synthesis of di-, T*T, tri-, T*T*T, and tetrameric, T*T*T*T, building blocks with carbamate internucleoside linkage, *=3′-NH-CO-O-5′, was achieved by the reaction of mono-, di-, or trimeric nucleoside 5′-O-p-nitrophenyl carbonate intermediates with 3′-deoxythymidine. These building blocks were suitably protected at 5′-position and converted into phosphoramidites, or attached onto CPG, and then used for the “chimeric oligonucleotide” synthesis. The novel oligonucleotides derived therefrom have been studied for their binding properties to complementary nucleic acids and for their nuclease sensitivity. Oligonucleotides containing one, two, or three carbamate linkages at 3′-end, were found to have increased nuclease resistance and did not effect the duplex stability significantly.